Biomarkers of response to nae inhibitors

ABSTRACT

Disclosed herein are markers whose mutational status is associated with sensitivity to treatment with NAE inhibitors. Mutational status is determined by measurement of characteristics of markers associated with the marker genes. Compositions and methods are provided to assess markers of marker genes to predict response to NAE inhibition treatment.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/552,686 filed on Oct. 28, 2011. The entire contents of the foregoingapplication are incorporated herein by reference.

SEQUENCE LISTING

This application contains a Sequence Listing which is submitted herewithin electronically readable format. The Sequence Listing file was createdon Oct. 26, 2012, is named “sequencelisting.txt,” and its size is 149 kb(153,088 bytes). The entire contents of the Sequence Listing in thesequencelisting.txt file are incorporated herein by this reference.

BACKGROUND

Cells become cancerous when their genotype or phenotype alters in a waythat there is uncontrolled growth that is not subject to the confines ofthe normal tissue environment. One or more genes is mutated, amplified,deleted, overexpressed or underexpressed. Chromosome portions can belost or moved from one location to another. Some cancers havecharacteristic patterns by which genotypes or phenotypes are altered.

Many genes have mutations which are associated with cancer. Some geneshave multiple sites where mutations can occur. Many cancers havemutations in and/or mis-expression of more than one gene. Gene mutationscan facilitate tumor progression, tumor growth rate or whether a tumorwill metastasize. Some mutations can affect whether a tumor cell willrespond to therapy.

A variety of agents treat cancers. Cancers of the blood and bone marrowoften are treated with steroids/glucocorticoids, imids, proteasomeinhibitors and alkylating agents. Cancers of other tissues often aretreated with alkylating agents, topoisomerase inhibitors, kinaseinhibitors, microtubule inhibitors, angiogenesis inhibitors or otheragents. Some patients respond to one therapy better than another,presenting the potential for a patient to follow multiple therapeuticroutes to effective therapy. Valuable time early in a patient'streatment program can be lost pursuing a therapy which eventually isproven ineffective for that patient. Many patients cannot afford thetime for trial-and-error choices of therapeutic regimens. Expedient andaccurate treatment decisions lead to effective management of thedisease.

SUMMARY

The present disclosure relates to prognosis and planning for treatmentof tumors by measurement of the amount, presence or changes of markersprovided herein. The markers are predictive of whether there will be afavorable outcome (e.g., good response, long time-to-progression, and/orlong term survival) after treatment with a NEDD8-activating enzyme (NAE)inhibitor, such as a 1-substituted methyl sulfamate. Testing samplescomprising tumor cells, e.g., in vitro, to determine the presence,amounts or changes of genetic markers, e.g., the mutational status of atleast one marker gene, identifies particular patients who are expectedto have a favorable outcome with treatment, e.g., with an NAE inhibitor,such as a 1-substituted methyl sulfamate, and whose disease may bemanaged by standard or less aggressive treatment, as well as thosepatients who are expected have an unfavorable outcome with the treatmentand may require an alternative treatment to, a combination of treatmentsand/or more aggressive treatment with an NAE inhibitor to ensure afavorable outcome and/or successful management of the disease.

In one aspect, the invention provides kits useful in determination ofcharacteristics, e.g., amounts, presence or changes, of the markers. Inanother aspect, the invention provides methods for determining prognosisand treatment or disease management strategies. In these aspects, thecharacteristic, e.g., size, sequence, composition or amount of marker ina sample comprising tumor cells is measured. In one embodiment, thetumor is a liquid, e.g., hematological tumor, e.g., acute myelogenousleukemia, myelodysplastic syndrome or multiple myeloma. In anotherembodiment, the tumor is a solid tumor, e.g., melanoma, non-small celllung cancer, esophageal cancer, bladder cancer, neuroblastoma cancer,mesothelioma, pancreatic cancer.

In various embodiments, the characteristic, e.g., size, sequence,composition or amount of DNA, the size, sequence, composition or amountof RNA and/or the size, sequence, composition or amount of proteincorresponding to a marker gene with one or more mutation, e.g., somaticmutation, described herein is measured. Useful information leading tothe prognosis or treatment or disease management strategies is obtainedwhen assays reveal information about a marker gene, e.g., whether thegene is mutated, or not, the identity of the mutation, and/or whetherthe RNA or protein amount of a mutated gene or genes indicatesoverexpression or underexpression. In one embodiment, the strategy isdetermined for E1 enzyme inhibition, e.g., NAE inhibition, e.g.,MLN4924, therapy.

A marker gene useful to test for determination of prognosis or treatmentor disease management strategy is selected from the group consisting ofneurofibromin 2 (NF2), mothers against decapentaplegic homolog 4(SMAD4), lysine-specific demethylase 6A (KDM6A), tumor protein p53(TP53), cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependentkinase inhibitor 2A p14 variant (CDKN2A_p14), in some cases, F-box andWD repeat domain containing 7 (FBXW7) and, in some cases adenomatouspolyposis coli (APC). Each marker gene includes mutations or alterationswhose presence in DNA or whose effects, e.g., on marker RNA and/orprotein characteristics, e.g., amounts, size, sequence or composition,can provide information for determination of prognosis or treatment ordisease management. In some embodiments, a gene or a mutant or modifiedform thereof useful as a marker, has a DNA, an RNA and/or proteincharacteristic, e.g., size, sequence, composition or amount, e.g., in asample comprising tumor cells, which is different than a normal DNA, RNAand/or protein. Described herein are examples of modifications of thesegenes, referred to as “marker genes” whose mutation or amounts canprovide such information.

The mutation of the markers of the present invention, provideinformation about outcome after treatment, e.g., with an NAE inhibitor,such as a 1-substituted methyl sulfamate. By examining thecharacteristic, e.g., size, sequence, composition or amount of one ormore of the identified markers in a tumor, it is possible to determinewhich therapeutic agent, combination of agents, dosing and/oradministration regimen is expected to provide a favorable outcome upontreatment. By examining the characteristic, e.g., size, sequence,composition or amount of one or more of the identified markers or markersets in a cancer, it is also possible to determine which therapeuticagent, combination of agents, dosing and/or administration regimen isless likely to provide a favorable outcome upon treatment. By examiningthe characteristic, e.g., size, sequence, composition or amount of oneor more of the identified markers, it is therefore possible to eliminateineffective or inappropriate therapeutic agents or regimens.Importantly, these determinations can be made on a patient-by-patientbasis. Thus, one can determine whether or not a particular therapeuticregimen is likely to benefit a particular patient or type of patient,and/or whether a particular regimen should be started or avoided,continued, discontinued or altered.

The present invention is directed to methods of identifying and/orselecting a cancer patient who is expected to demonstrate a favorableoutcome upon administration of a therapeutic regimen, e.g., atherapeutic regimen comprising an NAE inhibitor, such as a 1-substitutedmethyl sulfamate treatment. Additionally provided are methods ofidentifying a patient who is expected to have an unfavorable outcomeupon administration of such a therapeutic regimen. These methodstypically include measuring, determining, receiving, storing ortransmitting information about the characteristic, e.g., size, sequence,composition or amount of one or more markers or mutation of markergene(s) in a patient's tumor (e.g., a patient's cancer cells, e.g.,hematological cancer cells or solid tumor cells), optionally comparingthat to the characteristic, e.g., size, sequence, composition or amountof a reference marker, and in a further embodiment, identifying oradvising whether result from the sample corresponds to a favorableoutcome of a treatment regimen, e.g., an NAE inhibitor, such as a1-substituted methyl sulfamate treatment regimen.

Additionally provided methods include therapeutic methods which furtherinclude the step of beginning, continuing, or commencing a therapyaccordingly where the presence of a mutation in a marker gene or thecharacteristic, e.g., size, sequence, composition or amount of apatient's marker or markers indicates that the patient is expected todemonstrate a favorable outcome with the therapy, e.g., the NAEinhibitor, such as a 1-substituted methyl sulfamate therapeutic regimen.In addition, the methods include therapeutic methods which furtherinclude the step of stopping, discontinuing, altering or halting atherapy accordingly where the presence of a mutation in a marker gene orthe characteristic, e.g., size, sequence, composition or amount of apatient's marker indicates that the patient is expected to demonstratean unfavorable outcome with the treatment, e.g., with the NAE inhibitor,such as a 1-substituted methyl sulfamate regimen, e.g., as compared to apatient identified as having a favorable outcome receiving the sametherapeutic regimen. In another aspect, methods are provided foranalysis of a patient not yet being treated with a therapy, e.g., an NAEinhibitor, such as a 1-substituted methyl sulfamate therapy andidentification and prediction of treatment outcome based upon thepresence of a mutation in a marker gene or characteristic, e.g., size,sequence, composition or amount of one or more of a patient's markerdescribed herein. Such methods can include not being treated with thetherapy, e.g., NAE inhibitor, such as a 1-substituted methyl sulfamatetherapy, being treated with therapy, e.g., NAE inhibitor, or beingtreated with a 1-substituted methyl sulfamate therapy in combinationwith one more additional therapies, being treated with an alternativetherapy to an NAE inhibitor, such as a 1-substituted methyl sulfamatetherapy, or being treated with a more aggressive dosing and/oradministration regimen of a therapy, e.g., E1 enzyme inhibitor, such asan NAE inhibitor, e.g., as compared to the dosing and/or administrationregimen of a patient identified as having a favorable outcome tostandard NAE inhibitor, such as a 1-substituted methyl sulfamatetherapy. Thus, the provided methods of the invention can eliminateineffective or inappropriate use of therapy, e.g., NAE inhibitor, suchas 1-substituted methyl sulfamate therapy regimens.

Additional methods include methods to determine the activity of anagent, the efficacy of an agent, or identify new therapeutic agents orcombinations. Such methods include methods to identify an agent asuseful, e.g., as an NAE inhibitor, such as a 1-substituted methylsulfamate, for treating a cancer, e.g., a hematological cancer (e.g.,multiple myeloma, leukemias, lymphoma, etc) or solid tumor cancer (e.g.,melanoma, esophageal cancer or bladder cancer), based on its ability toaffect the presence of a mutation in a marker gene or characteristic,e.g., size, sequence, composition or amount of a marker or markers ofthe invention. For example, an inhibitor which decreases or increasesthe presence of a mutation in a marker gene or characteristic, e.g.,size, sequence, composition or amount of a marker or markers provided ina manner that indicates favorable outcome of a patient having cancerwould be a candidate agent for the cancer. Alternatively, an agent whichis able to decrease the viability of a tumor cell comprising a markerindicative of an unfavorable outcome would be a candidate agent for thecancer.

The present invention is also directed to methods of treating a cancerpatient, with a therapeutic regimen, e.g., an NAE inhibitor, such as a1-substituted methyl sulfamate therapy regimen (e.g., alone, or incombination with an additional agent such as a chemotherapeutic agent,e.g., a glucocorticoid agent, a proteasome inhibitor, an alkylatingagent, a kinase inhibitor or a topoisomerase inhibitor), which includesthe step of selecting for treatment a patient whose markercharacteristic, e.g., size, sequence, composition or amount indicatesthat the patient is expected to have a favorable outcome with thetherapeutic regimen, and treating the patient with the therapy, e.g.,NAE inhibition, such as a 1-substituted methyl sulfamate therapy. Insome embodiments, the method can include the step of selecting a patientwhose marker characteristic, e.g., size, sequence, composition or amountor amounts indicates that the patient is expected have a favorableoutcome and administering a therapy other than an NAE inhibitor therapythat demonstrates similar expected survival times as the NAE inhibitor,such as a 1-substituted methyl sulfamate therapy.

Additional methods of treating a cancer patient include selectingpatients that are unlikely to experience a favorable outcome upontreatment with a cancer therapy (e.g., NAE inhibitor, such as a1-substituted methyl sulfamate therapy). Such methods can furtherinclude one or more of: administering a higher dose or increased dosingschedule of a therapy, e.g., NAE inhibitor, such as a 1-substitutedmethyl sulfamate as compared to the dose or dosing schedule of a patientidentified as having a favorable outcome with standard therapy;administering a cancer therapy other than an NAE inhibitor, such as a1-substituted methyl sulfamate therapy; administering an NAE inhibitor,such as a 1-substituted methyl sulfamate agent in combination with anadditional agent. Further provided are methods for selection of apatient having aggressive disease which is expected to demonstrate morerapid time to progression and death.

Additional methods include a method to evaluate whether to treat or payfor the treatment of cancer, e.g., hematological cancer (e.g., multiplemyeloma, leukemias, lymphoma, etc.) or solid tumor cancer (e.g.,melanoma, esophageal cancer or bladder cancer) by reviewing the amountof a patient's marker or markers for indication of outcome to a cancertherapy, e.g., an NAE inhibitor, such as a 1-substituted methylsulfamate therapy regimen, and making a decision or advising on whetherpayment should be made.

The entire contents of all publications, patent applications, patentsand other references mentioned herein are incorporated by reference.

Other features and advantages of the invention will be apparent from thefollowing detailed description, drawings and from the claims.

DRAWINGS

FIG. 1. General structure of 1-substituted methyl sulfamate. G¹ is —O—or —CH₂—; G² is —H or —OH; G³ is —H or —OH; G⁴ is —NH—, —O— or acovalent bond; and G⁵ is substituted heteroaryl.

FIG. 2. General pathways for cullin-ring ligase (CRL) ubiquitination ofprotein substrates and for neddylation. In CRLs, the cullin subunit mustbe modified on a conserved lysine by the ubiquitin-like protein NEDD8 toactivate holoenzyme activity. NEDD8 activation and conjugation to cullinproteins is catalyzed via an enzymatic cascade that is homologous toubiquitination involving NEDD8's E1 (NAE) and E2 (Ubc12). Removal ofNEDD8 from cullin is catalyzed by the COP9 signalosome. Deneddylationfacilitates dissociation of CRL components. The cullin-RING core issequestered in an inactive state by binding to CAND1 until it isrecruited to form a new CRL.

FIG. 3. Response of a cell line panel 2 to MLN4924. Each pointrepresents one cell line.

FIGS. 4A-B. Comparison of responses of cell line panels to MLN4924. A.Ordering of cell line panel 2 by EC50. Darkened lines represent celllines that are present in panel 1. There are 114 cell lines withidentical names in both panels. B. Comparison of Percent of Control(POC) viability for the cell lines which are present in both panels. Theresults of the overlapping cell lines have a Spearman Rank OrderCorrelation of 0.72, p-value<2.2e-16.

FIG. 5. Tissue association of resistance with TP53 mutations. TP53mutant colon cancer cell lines are more resistant (higher percent ofcontrol viability) to MLN4924 treatment than TP53 wt cell lines.

FIGS. 6A-D. Effect of TP53 Loss on Viability of Cancer Cell LinesFollowing Treatment with Different Doses of MLN4924 at Multiple TimePoints. The effect of TP53 knock-out on the sensitivity of pairedHCT-116 colon cancer cell lines to MLN4924 was measured by ATPlite,across a range of MLN4924 concentrations and time points. Data arerepresented as mean±SEM, N=3. Dotted line, p53 knock-out; solid line,p53 wild type.

DETAILED DESCRIPTION

One of the continued problems with therapy in cancer patients isindividual differences in response to therapies. While advances indevelopment of successful cancer therapies progress, only a subset ofpatients respond to any particular therapy. With the narrow therapeuticindex and the toxic potential of many available cancer therapies, suchdifferential responses potentially contribute to patients undergoingunnecessary, ineffective and even potentially harmful therapy regimens.If a designed therapy could be optimized to treat individual patients,such situations could be reduced or even eliminated. Furthermore,targeted designed therapy may provide more focused, successful patienttherapy overall. Accordingly, there is a need to identify particularcancer patients who are expected to have a favorable outcome whenadministered particular cancer therapies as well as particular cancerpatients who may have a favorable outcome using more aggressive and/oralternative cancer therapies, e.g., alternative to previous cancertherapies administered to the patient. It would therefore be beneficialto provide for the diagnosis, staging, prognosis, and monitoring ofcancer patients, including, e.g., hematological cancer patients (e.g.,multiple myeloma, leukemias, lymphoma, etc.) or solid tumor cancer(e.g., melanoma, esophageal cancer or bladder cancer) who would benefitfrom particular cancer inhibition therapies as well as those who wouldbenefit from a more aggressive and/or alternative cancer inhibitiontherapy, e.g., alternative to a cancer therapy or therapies the patienthas received, thus resulting in appropriate preventative measures.

The present invention is based, in part, on the recognition thatmutation of a marker gene can be associated with sensitivity of a cellcomprising the mutated gene to an NAE inhibitor, such as a 1-substitutedmethyl sulfamate. In some embodiments, the marker gene is involved inthe cullin ring ligase (CRL) pathway, e.g., a gene whose encoded proteininteracts with a CRL or a CRL-associated protein, or is a CRL substrate.A protein encoded by a marker gene can have a wild type function as atumor suppressor. Examples of marker genes include NF2, SMAD4 and/orKDM6A. Other examples of marker genes include TP53, APC, CDKN2A and/orCDKN2A_p14. Marker genes can exhibit mutations, e.g., somatic mutations,whose presence can affect expression or activity of the encoded geneproduct. In some embodiments, there can be more than one mutation in amarker gene or more than one marker gene with a mutation in a tumor cellor tumor. In additional embodiments, there can be marker gene mutationsin cells which have mutations in additional genes, including mutationsthat can lead to tumorigenesis, but the additional mutated genes may notbe marker genes as considered herein. In some embodiments, the mutationis an inactivating mutation. In other embodiments, the mutation affectsthe expression of the marker gene. In other embodiments, a mutation canresult in an altered interaction of the encoded gene product with acellular binding partner. The identification and/or measurement of themutation in the marker gene can be used to determine whether a favorableoutcome can be expected by treatment of a tumor, e.g., with an NAEinhibitor, such as a 1-substituted methyl sulfamate therapy or whetheran alternative therapy to and/or a more aggressive therapy with, e.g.,an NAE inhibitor, such as a 1-substituted methyl sulfamate inhibitor mayenhance expected survival time. For example, the compositions andmethods provided herein can be used to determine whether a patient isexpected to have a favorable outcome to an NAE inhibitor, such as a1-substituted methyl sulfamate therapeutic agent or an NAE inhibitor,such as a 1-substituted methyl sulfamate dosing or administrationregimen. In general, mutation in the tumor suppressor marker genesdescribed herein is associated with sensitivity to or favorable outcomeof treatment with a NAE inhibitor. Examples of marker genes which canfunction as a tumor suppressor in pathways related to cullin ring ligaseand whose mutation is associated with sensitivity to NAE inhibitioninclude NF2, SMAD4, KDM6A, FBXW7, CDKN2A and/or CDKN2A_p14. However,TP53 and APC also are tumor suppressor marker genes. In particular, TP53pathway genes are associated with NAE inhibitor effects. As describedherein, in some embodiments for many tumor types, mutation in TP53, andin some cases, APC, leads to resistance to NAE inhibition. Accordingly,a wild type marker gene from the group consisting TP53 and APC can beassociated with NAE sensitivity. In some embodiments, mutation of amarker gene selected from the group consisting of TP53 and APC isassociated with resistance to an NAE inhibitor.

Based on these identifications, the present invention provides, withoutlimitation: 1) methods and compositions for determining whether an NAEinhibitor, such as a 1-substituted methyl sulfamate therapy regimen willor will not be effective to achieve a favorable outcome and/or managethe cancer; 2) methods and compositions for monitoring the effectivenessof an NAE inhibitor, such as a 1-substituted methyl sulfamate therapy(alone or in a combination of agents) and dosing and administrationsused for the treatment of tumors; 3) methods and compositions fortreatments of tumors comprising, e.g., NAE inhibitor, such as a1-substituted methyl sulfamate inhibition therapy regimen; 4) methodsand compositions for identifying specific therapeutic agents andcombinations of therapeutic agents as well as dosing and administrationregimens that are effective for the treatment of tumors in specificpatients; and 5) methods and compositions for identifying diseasemanagement strategies.

Ubiquitin and other ubiquitin-like molecules (ubls) are activated by aspecific enzyme (an E1 enzyme) which catalyzes the formation of anacyl-adenylate intermediate with the C-terminal glycine of the ubl. Theactivated ubl is then transferred to a catalytic cysteine residue withinthe E1 enzyme through formation of a thioester bond intermediate. TheE1-ubl intermediate and an E2 associate, resulting in a thioesterexchange wherein the ubl is transferred to the active site cysteine ofthe E2. The ubl is then conjugated to the target protein, eitherdirectly or in conjunction with an E3 ligase, through isopeptide bondformation with the amino group of a lysine side chain in the targetprotein. The ubl named Neural precursor cell-Expressed DevelopmentallyDownregulated 8 (NEDD8) is activated by the heterodimer NEDD8-activatingenzyme (NAE, also known as APPBP1-UBA3, UBE1C (ubiquitin-activatingenzyme E1C)) and is transferred to one of two E2 conjugating enzymes(ubiquitin carrier protein 12 (UBC12) and UBC17), ultimately resultingin ligation of NEDD8 to cullin proteins by the cullin-RING subtype ofubiquitin ligases (see FIG. 2). A function of neddylation is theactivation of cullin-based ubiquitin ligases involved in the turnover ofmany cell cycle and cell signaling proteins, including p27 and I-κB. SeePan et al., Oncogene 23:1985-97 (2004). Inhibition of NAE can disruptcullin-RING ligase-mediated protein turnover and can lead to apoptoticdeath in cells, e.g., tumor cells or cells of a pathogenic organism,e.g. a parasite. See Soucy et al. (2010) Genes & Cancer 1:708-716.

As used herein, the term “E1,” “E1 enzyme,” or “E1 activating enzyme”refers to any one of a family of related ATP-dependent activatingenzymes involved in activating or promoting ubiquitin or ubiquitin-like(collectively “ubl”) conjugation to target molecules. E1 activatingenzymes function through an adenylation/thioester intermediate formationto transfer the appropriate ubl to the respective E2 conjugating enzymethrough a transthiolation reaction. The resulting activated ubl-E2promotes ultimate conjugation of the ubl to a target protein. A varietyof cellular proteins that play a role in cell signaling, cell cycle, andprotein turnover are substrates for ubl conjugation which is regulatedthrough E1 activating enzymes (e.g., NAE, UAE, SAE). Unless otherwiseindicated by context, the term “E1 enzyme” is meant to refer to any E1activating enzyme protein, including, without limitation, NEDD8activating enzyme (NAE (APPBP1/Uba3)), ubiquitin activating enzyme (UAE(Uba1)), sumo activating enzyme (SAE (Aos1/Uba2)), UBA4, UBA5, UBA6,ATG7 or ISG15 activating enzyme (Ube1L).

The term “E1 enzyme inhibitor” or “inhibitor of E1 enzyme” is used tosignify a compound having a structure as defined herein, which iscapable of interacting with an E1 enzyme and inhibiting its enzymaticactivity. Inhibiting E1 enzymatic activity means reducing the ability ofan E1 enzyme to activate ubiquitin like (ubl) conjugation to a substratepeptide or protein (e.g., ubiquitination, neddylation, sumoylation). Insome embodiments, an E1 enzyme inhibitor can inhibit more than one E1enzyme. In other embodiments, an E1 enzyme inhibitor is specific for aparticular E1 enzyme. In various embodiments, such reduction of E1enzyme activity is at least about 50%, at least about 75%, at leastabout 90%, at least about 95%, or at least about 99%. In variousembodiments, the concentration of E1 enzyme inhibitor required to reducean E1 enzymatic activity is less than about 1 μM, less than about 500nM, less than about 100 nM, less than about 50 nM, or less than about 10nM.

As used herein, the term “NAE inhibitor” refers to an inhibitor of theNAE heterodimer. Examples of NAE inhibitors include 1-substituted methylsulfamates (see FIG. 1), including MLN4924. Langston S. et al. U.S.patent application Ser. No. 11/700,614, whose PCT application waspublished as WO07/092213, WO06084281 and WO2008/019124 (the entirecontents of each of the foregoing published patent applications arehereby incorporated by reference), disclose compounds which areeffective inhibitors of E1 activating enzymes, e.g., NAE. In someembodiments, NAE inhibitors do not inhibit, or are very poor atinhibiting, other (non-NAE) E1 enzymes. The compounds are useful forinhibiting E1 activity in vitro and in vivo and are useful for thetreatment of disorders of cell proliferation, e.g., cancer, and otherdisorders associated with E1 activity, such as pathogenic infections andneurodegenerative disorders. One class of compounds described inLangston et al. are 4-substituted((1S,2S,4R)-2-hydroxy-4-{7H-pyrrolo[2,3-d]pyrimidin-7-yl}cyclopentyl)methylsulfamates.

MLN4924(((1S,2S,4R)-4-{4-[(1S)-2,3-dihydro-1H-inden-1-ylamino]-7H-pyrrolo[2,3-d]pyrimidin-7-yl}-2-hydroxycyclopentyl)methylsulphamate) is an NAE-specific E1 inhibitor which disrupts cullin-RINGligase-mediated protein turnover leading to apoptotic death in humantumor cells by perturbation of cellular protein homeostasis (Soucy etal. (2009) Nature 458:732-736). The evaluation of MLN4924 in cellularand tumor xenograft studies has revealed two distinct mechanisms ofaction. The first is the induction of DNA re-replication, DNA damage andcell death through MLN4924-mediated dysregulation of the CRL1^(SKP)2 andCRL4^(DDB1) substrate Cdt-1 (Milhollen et al. (2011) Cancer Res.71:3042-3051). It has been shown that p53 status does not impact theinduction of DNA re-replication but may make cells more prone to undergoapoptosis or senescence depending on the appropriate genetic background(Milhollen et al. (2011) supra, Lin et al. (2010) Nature 464:374-379 andLin et al. (2010) Cancer Res. 70:10310-20). The second mechanism is theinhibition of NF-κB pathway activity in NF-κB dependent Diffuse LargeB-Cell Lymphomas primarily through dysregulation of CRL1^(βTRCP)mediated turnover of phosphorylated IκBα (Milhollen et al. (2010) Blood116:1515-1523). In addition, pre-clinical models of Acute MyelogenousLeukemia (AML) are sensitive to MLN4924 inhibition in both cell linesand primary patient blasts through mechanisms related to Cdt-1dysregulation, NF-κB inhibition and induction of reactive oxygen species(Swords et al. (2010) Blood 115:3796-3800).

Genes such as NF2 (reviewed by Ahronowitz et al. (2007) Human Mutation28:1-2), KDM6A (reviewed by van Haaften et al. (2009) Nat. Genet.41:521-523), FBXW7, TP53, CDKN2A and CDKN2A_p14 are mutated in manycancer types. SMAD4 is mutated in a number of cancers, but many of SMAD4mutations are found in cancers of the intestine, pancreas (reviewed byMiyaki and Kuroki (2003) Biochem. Biophys. Res. Commun. 306:799-804) orthyroid gland.

As used herein, “NF2” refers to the longer isoform of the geneassociated with GenBank Accession No. NM_(—)000268, SEQ ID NO:1 (openreading frame is SEQ ID NO:2, nucleotides 444 to 2231 of SEQ ID NO:1),encoding GenPept Accession No. NP_(—)000259, SEQ ID NO:3). Other namesfor NF2 include ACN, BANF, SCH and merlin (moesin-ezrin-radixin-likeprotein). NF2 functions as a tumor suppressor gene and can be found onchromosome 22. NF2 interacts with the cytoskeleton, cell surfaceproteins and may be involved in cytoskeletal dynamics and regulating iontransport. Functions of NF2 that can relate it to sensitivity to NAEinhibition, e.g., MLN4924 include its ability to inhibit the E3ubiquitin ligase CRL4^(DCAF1) (Li et al. (2010) Cell 140:477-490).Mutations in NF2 can disrupt its inhibitory activity and lead touncontrolled ubiquitination of substrates of CRL4^(DCAF1) andproliferation of cells harboring the mutated gene.

As used herein, “SMAD4” refers to the gene associated with GenBankAccession No. NM_(—)005359, SEQ ID NO:4 (open reading frame is SEQ IDNO:5, nucleotides 539 to 2197 of SEQ ID NO:4), encoding GenPeptAccession No. NP_(—)005350, SEQ ID NO:6. Other names for SMAD4 includedeleted in pancreatic carcinoma locus 4 (DPC4), JIP, or mothers againstdecapentaplegic, Drosophila, homolog of, 4 (MAD4). SMAD4 is a signaltransduction protein involved in transforming growth factor (TGF)-betasignaling. SMAD4 can act as a tumor suppressor and can be targeted fordegradation by ubiquitination by the Skp-Cullin-F-box protein (SCF)complex.

As used herein, “KDM6A” refers to the gene associated with GenBankAccession No. NM_(—)021140, SEQ ID NO:7 (open reading frame is SEQ IDNO:8, nucleotides 376 to 4581 of SEQ ID NO:7, or SEQ ID NO:9), encodingGenPept Accession No. NP_(—)066963, SEQ ID NO:10 or SEQ ID NO:11 (SEQ IDNO:10 with a V instead of an L at position 173 and an R instead of L at584, an N instead of S at position 601 and/or K instead of E at position629). Other names for KDM6A include ubiquitously-transcribedtetratricopeptide repeat protein X-linked or ubiquitously-transcribedTPR gene on the X chromosome (UTX), or bA286N14.2. KDM6A is a histonedemethylase and can function as a tumor suppressor.

As used herein, “FBXW7” refers to the gene associated with GenBankAccession No. NM_(—)033632, SEQ ID NO:12 (open reading frame is SEQ IDNO:13, nucleotides 150 to 2273 of SEQ ID NO:12), encoding GenPeptAccession No. NP_(—)361014, SEQ ID NO:14. Other names for FBXW7 includehomolog of C. elegans sel-10 (SEL10), archipelago homolog (AGO), F-boxprotein FBX30 (FBXO30), or cell division control protein 4 (CDC4). FBXW7can associate into a ubiquitin protein ligase complex to participate inphosphorylation-dependent ubiquitination of proteins, including proteinsinvolved in cell cycle and survival. FBXW7 can act as a tumorsuppressor. Use of FBXW7 as marker gene may be organ-specific, i.e., itcan be a marker of sensitivity in tumors arising in some tissues but notothers. For example, FBXW7 can be a marker of sensitivity in tumors ofthe uterus, cervix or liver, but not a marker of sensitivity in tumorsof the digestive tract, where mutations in other genes may dominate toresult in the insensitivity or resistance of cells from those tumors toMLN4924.

As used herein, “TP53” refers to the gene associated with GenBankAccession No. NM_(—)000546, SEQ ID NO:15 (open reading frame is SEQ IDNO:16, nucleotides 203 to 1384 of SEQ ID NO:15, or a variant wherein thenucleotide at position 417 is a guanine instead of a cytosine), encodingGenPept Accession No. NP_(—)000537, SEQ ID NO:17 or a variant whereinthe amino acid residue at position 72 is an arginine, R instead of aproline, P). Other names for TP53 include BCC7, LFS1 and p53. TP53 bindsDNA and activates transcription factors and can function as a tumorsuppressor.

As used herein, “CDKN2A” refers to the gene associated with GenBankAccession No. NM_(—)000077, SEQ ID NO:18 (open reading frame is SEQ IDNO:19, nucleotides 307 to 777 of SEQ ID NO:18), encoding GenPeptAccession No. NP_(—)000068, SEQ ID NO:20. Other names for CDKN2A includealternate open reading frame (ARF), p16, p16ARF, inhibitor ofcyclin-dependent kinase 4 (INK4) and multiple tumor suppressor gene-1(MTS1). Variants of CDKN2A differ in the first exon. One variant is“CDKN2A_p14” or “CDKN2A.p14,” also known as p14ARF, is associated withGenBank accession number NM_(—)058195, SEQ ID NO:21 (open reading frameis SEQ ID NO:22, nucleotides 38 to 559 of SEQ ID NO:21); GenPeptNP_(—)478102, SEQ ID NO:23 or a variant which begins at amino acidresidue 42 of SEQ ID NO:23. CDKN2A_p14 results from translation in adifferent reading frame than p16ARF (p16INK4a, CDKN2A). CDKN2A andCDKN2A_p14 inhibit cyclin dependent kinase 4, can stabilize p53 and canregulate cell cycle G1 progression. CDKN2A and CDKN2A_p14 can act as atumor suppressor.

As used herein, “APC” refers to adenomatous polyposis coli, the geneassociated with GenBank Accession No. NM_(—)000038, SEQ ID NO:24 (openreading frame SEQ ID NO:25, or a variant with a thymine instead of acytosine at nucleotide 1458), encoding GenPept Accession No.NP_(—)000029, SEQ ID NO:26. Other names for APC include BTSP2, and DP2.APC binds microtubules and inhibits the Wnt-signalling pathway and canfunction as a tumor suppressor.

There has been interest in public cataloging mutations associated withcancers. Examples of public databases which include information aboutmutations associated with cancers are the Database of Genotypes andPhenotypes (dbGaP) maintained by the National Center for BiotechnologyInformation (Bethesda, Md.) and Catalogue of Somatic Mutations in Cancer(COSMIC) database maintained by the Wellcome Trust Sanger Institute(Cambridge, UK).

Compositions and methods are provided to determine the mutationalstatus, e.g., to identify mutations in marker genes in hematological(e.g., multiple myeloma, leukemias, lymphoma, etc.) or solid (e.g.,melanoma, esophageal cancer, lung cancer or bladder cancer) tumors topredict response to treatment, time-to-progression and survival upontreatment. Compositions and methods provided herein also can identifymutations in marker genes in solid tumors such as from colon cancer,breast cancer, head and neck cancer, or central nervous system cancer.

Markers were identified based on genetic profiles of tumor cells whichexhibit sensitivity to treatment to MLN4924. TP53 marker also wasidentified based on the behavior of isogenic cell lines which differ inthe deletion of the TP53 gene. Observed sensitivity can be consistentamong tumor cells tested by more than one method.

Unless otherwise defined, all technical and scientific terms used hereinhave the meanings which are commonly understood by one of ordinary skillin the art to which this invention belongs. Generally, nomenclatureutilized in connection with, and techniques of cell and tissue culture,molecular biology and protein and oligo- or polynucleotide chemistry andhybridization described herein are those known in the art. GenBank orGenPept accession numbers and useful nucleic acid and peptide sequencescan be found at the website maintained by the National Center forBiotechnology Information, Bethesda, Md. The content of all databaseaccession records (e.g., from Affymetrix HG133 annotation files, Entrez,GenBank, RefSeq, COSMIC) cited throughout this application (includingthe Tables) are hereby incorporated by reference. Standard techniquesare used for recombinant DNA, oligonucleotide synthesis, proteinpurification, tissue culture and transformation and transfection (e.g.,electroporation, lipofection, etc). Enzymatic reactions are performedaccording to manufacturer's specifications or as commonly accomplishedin the art or as described herein. The foregoing techniques andprocedures generally are performed according to methods known in theart, e.g., as described in various general and more specific referencesthat are cited and discussed throughout the present specification. Seee.g., Sambrook et al. (2000) Molecular Cloning: A Laboratory Manual(3^(rd) ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.) or Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Thenomenclatures utilized in connection with, and the laboratory proceduresand techniques of, analytical chemistry, synthetic organic chemistry,and medicinal and pharmaceutical chemistry described herein are known inthe art. Standard techniques are used for chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation and delivery, andtreatment of patients. Furthermore, unless otherwise required bycontext, singular terms shall include pluralities and plural terms shallinclude the singular. In the case of conflict, the presentspecification, including definitions, will control.

The articles “a,” “an” and “at least one” are used herein to refer toone or to more than one of the grammatical object of the article. By wayof example, “an element” means one or more than one element, at leastone element. In the case of conflict, the present specification,including definitions, will control.

As used herein, a “favorable” outcome or prognosis refers to long termsurvival, long time-to-progression (TTP), and/or good response.Conversely, an “unfavorable” prognosis refers to short term survival,short time-to-progression (TTP) and/or poor response.

A “marker” as used herein, includes a material associated with a markergene which has been identified as having a mutation in tumor cells of apatient and furthermore that mutation is characteristic of a patientwhose outcome is favorable or unfavorable with treatment e.g., by an NAEinhibitor, such as a 1-substituted methyl sulfamate. Examples of amarker include a material, e.g., a chromosome locus, DNA for a gene, RNAfor a gene or protein for a gene. For example, a marker includes amarker gene material, e.g., a chromosome locus, DNA, RNA or proteinwhich demonstrates a characteristic, e.g., size, sequence, compositionor amount indicative of a short term survival patient; alternatively amarker includes a marker gene material, e.g., a chromosome locus, DNA,RNA or protein which demonstrates a mutation or characteristic, e.g.,size, sequence, composition or amount indicative of a long term survivalpatient. In another example, a marker includes a marker gene material,e.g., a chromosome locus, DNA, RNA or protein whose mutation orcharacteristic, e.g., size, sequence, composition or amount isindicative of a patient with a poor response to treatment; alternativelya marker includes a marker gene material, e.g., a chromosome locus, DNA,RNA or protein whose mutation or characteristic, e.g., size, sequence,composition or amount is indicative of a patient with a good response.In a further example, a marker includes a marker gene material, e.g., achromosome locus, DNA, RNA or protein whose mutation or characteristic,e.g., size, sequence, composition or amount is indicative of a patientwhose disease has a short time-to-progression (TTP) upon treatment;alternatively a marker includes a marker gene material, e.g., achromosome locus, DNA, RNA or protein whose mutation or characteristic,e.g., size, sequence, composition or amount is indicative of a patientwhose disease has a long TTP. In a yet a further example, a markerincludes a marker gene material, e.g., a chromosome locus, DNA, RNA orprotein whose mutation or characteristic, e.g., size, sequence,composition or amount is indicative of a patient whose disease has ashort term survival upon treatment; alternatively a marker includes amarker gene material, e.g., a chromosome locus, DNA, RNA or proteinwhose mutation or characteristic, e.g., size, sequence, composition oramount is indicative of a patient whose disease has a long termsurvival. Thus, as used herein, marker is intended to include each andevery one of these possibilities, and further can include each singlemarker individually as a marker; or alternatively can include one ormore, or all of the characteristics collectively when reference is madeto “markers” or “marker sets.”

A chromosome locus marker useful to measure for determination ofprognosis or treatment or disease management strategy is selected fromthe group consisting of chromosome 22q12.2 (NF2), e.g., from base pair29999545 to 30094589, chromosome 18q21.1-21.2 (SMAD4), e.g., from basepair 48556583 to 48611412, chromosome Xp11.2 (KDM6A), e.g., from basepair 44732423 to 44971847, chromosome 4q31.3 (FBXW7), e.g., from basepair 153242410-153456172, chromosome 17p13.1 (TP53), e.g., from basepair 7571720 to 7590868, and 9p21 (CDKN2A and CDKN2A_p14), e.g., frombase pair 21967751 to 21994490. Chromosome locus numbers are based onthe reference human genome Build 37.3 (current as of Oct. 5, 2011) inthe NCBI Gene database. A marker DNA, marker RNA or marker protein cancorrespond to base pairs on a chromosome locus marker. For example, amarker DNA can include genomic DNA from a chromosome locus marker,marker RNA can include a polynucleotide transcribed from a locus marker,and a marker protein can include a polypeptide resulting from expressionat a chromosome locus marker in a sample, e.g., comprising tumor cells.

A “marker nucleic acid” is a nucleic acid (e.g., genomic DNA, mRNA,cDNA) encoded by or corresponding to a marker gene of the invention.Such marker nucleic acids include DNA, e.g., sense and anti-sensestrands of genomic DNA (e.g., including any introns occurring therein),comprising the entire or a partial sequence, e.g., one or more of theexons of the genomic DNA, up to and including the open reading frame ofany of the marker genes or the complement of such a sequence. The markernucleic acids also include RNA comprising the entire or a partialsequence of any marker or the complement of such a sequence, wherein allthymidine residues are replaced with uridine residues, RNA generated bytranscription of genomic DNA (i.e. prior to splicing), RNA generated bysplicing of RNA transcribed from genomic DNA, and proteins generated bytranslation of spliced RNA (i.e. including proteins both before andafter cleavage of normally cleaved regions such as transmembrane signalsequences). As used herein, a “marker nucleic acid” may also include acDNA made by reverse transcription of an RNA generated by transcriptionof genomic DNA (including spliced RNA). A marker nucleic acid alsoincludes sequences which differ, due to degeneracy of the genetic code,from the nucleotide sequence of nucleic acids encoding a protein whichcorresponds to a marker, e.g., a mutated marker, of the invention, andthus encode the same protein, e.g., mutated protein. As used herein, thephrase “allelic variant” refers to a nucleotide sequence which occurs ata given locus or to a polypeptide encoded by the nucleotide sequence.Such naturally occurring allelic variations can typically result in 1-5%variance in the nucleotide sequence of a given gene. Alternative allelescan be identified by sequencing the gene of interest in a number ofdifferent individuals, e.g., in cells, e.g., germline cells, ofindividuals without cancer. This can be readily carried out by usinghybridization probes to identify the same genetic locus in a variety ofindividuals. Detection of any and all such nucleotide variations andresulting amino acid polymorphisms or variations that are the result ofnaturally occurring allelic variation and that do not alter thefunctional activity of a wild type marker gene is intended to be withinthe scope of the wild type version of a marker described herein. A“marker protein” is a protein encoded by or corresponding to a marker,e.g., a mutant nucleic acid, of the invention. The terms “protein” and“polypeptide” are used interchangeably. A protein of a markerspecifically can be referred to by its name or amino acid sequence, butit is understood by those skilled in the art, that mutations, deletionsand/or post-translational modifications can affect protein structure,appearance, cellular location and/or behavior. Unless indicatedotherwise, such differences are not distinguished herein, and a markerdescribed herein is intended to include any or all such varieties.

As used herein, a “marker gene” refers to a gene which can have amutation such that its DNA, RNA and/or protein has a characteristic,e.g., size, sequence, composition or amount(s) which provide informationabout prognosis (i.e., are “informative”) upon treatment. Marker genesdescribed herein as linked to outcome after NAE inhibitor, such as1-substituted methyl sulfamate (e.g., MLN4924) treatment are examples ofgenes within the chromosome locus markers described above and areprovided in Table 1. Sequences of mRNA, open reading frames and proteinscorresponding to marker genes also are listed in Table 1. A marker genelisted in Table 1 can have isoforms which are either ubiquitous or haverestricted expression. Except for the separate listing of the CDKN2A_p14isoform, the DNA SEQ ID NOs in Table 1 refer to the mRNA encoding themajor or longest isoform and the protein SEQ ID NOs represent at least aprecursor of such isoform and not necessarily the mature protein. Thesesequences are not intended to limit the marker gene identity to thatisoform or precursor. The additional isoforms and mature proteins arereadily retrievable and understandable to one of skill in the art byreviewing the information provided under the Entrez Gene (databasemaintained by the National Center for Biotechnology Information,Bethesda, Md.) identified by the ID number listed in Table 1.

TABLE 1 Marker Gene Description for NAE Inhibitor Treatment Chromo-Marker Gene Entrez some Start base End base SEQ ID ID Marker Gene NameGene ID location pair pair NOs: NF2 neurofibromin 2 4771 22q 2999954530094589 1, 2, 3 SMAD4 mothers against 4089 18q 48556583 48611412 4, 5,6 decapentaplegic homolog 4 KDM6A lysine-specific 7403 Xp 4473242344971847 7, 8, 9, demethylase 6A 10, 11 FBXW7 F-box and WD 55294 4q153242410 153456172 12, 13, 14 repeat domain containing 7 TP53 tumorprotein p53 7157 17p 7571720 7590868 15, 16, 17 CDKN2A cyclin-dependent1029 9p 21967751 21994490 18, 19, 20 kinase inhibitor 2A CDKN2A_p14cyclin-dependent 1029 9p 21967751 21994490 21, 22, 23 kinase inhibitor2A p14 variant APC adenomatous 324 5q 112043202 112181936 24, 25, 26polyposis coli

As used herein, an “informative” characteristic, e.g., size, sequence,composition or amount of a marker refers to a characteristic, e.g.,size, sequence, composition or amount whose value or difference iscorrelated to prognosis or outcome. The informative characteristic,e.g., size, sequence, composition or amount of a marker can be obtainedby analyzing either nucleic acid, e.g., DNA or RNA, or proteincorresponding to the marker gene. The characteristic, e.g., size (e.g.,length or molecular weight), sequence (e.g., nucleic acid sequence orprotein sequence), composition (e.g., base or amino acid composition orpeptide digest or gene fragment pattern) or amount (e.g., copy numberand/or expression level) of a marker, e.g., a chromosome locus marker ora marker in a sample from a patient can be “informative” if it isdifferent than the wild type or allelic variant of the substance beinganalyzed. In some embodiments, a characteristic of a marker isinformative if it indicates that the marker gene is wild type. In anembodiment where the amount of a marker is being measured, an amount is“informative” if it is greater than or less than a reference amount by adegree greater than the standard error of the assay employed to assessexpression. The informative expression level of a marker can bedetermined upon statistical correlation of the measured expression leveland the outcome, e.g., good response, poor response, longtime-to-progression, short time-to-progression, short term survival orlong term survival. The result of the statistical analysis can establisha threshold for selecting markers to use in the methods describedherein. Alternatively, a marker, e.g., a chromosome locus marker, or amarker gene that has differential characteristic, e.g., size, sequence,composition or amounts will have typical ranges of amounts that arepredictive of outcome. An informative characteristic, e.g., size,sequence, composition or amount is a characteristic, e.g., size,sequence, composition or amount that falls within the range ofcharacteristic, e.g., size, sequence, composition or amounts determinedfor the outcome. Still further, a set of markers may together be“informative” if the combination of their characteristics, e.g., sizes,sequences, compositions or amounts either meets or is above or below apre-determined score for a marker, e.g., a chromosome locus marker, or amarker gene, set as determined by methods provided herein. Genetranslocation, transcript splice variation, deletion and truncation areexamples of events which can change marker size, sequence orcomposition, in addition to point mutations which can change markersequence or composition. Measurement of only one characteristic, e.g.,marker, of a marker gene (i.e., DNA, RNA or protein) can provide aprognosis, i.e., indicate outcome. Measurement of more than onecharacteristic, e.g., marker, of a marker gene can provide a prognosiswhen the informative amounts of the two characteristics are consistentwith each other, i.e., the biologies of the results are notcontradictory. Examples of consistent results from measurement ofmultiple characteristics of a marker gene can be identification of anonsense mutation or deletion in a DNA or RNA and a low amount or lowmolecular weight of encoded protein, or a mutation in a region whichencodes a binding pocket or active site of a protein and low activity ofthe encoded protein. A different example can occur when a protein is ina pathway with a feedback loop controlling its synthesis based on itsactivity level. In this example, a low amount or activity of protein canbe associated with a high amount of its mutated mRNA as a tissue, due tothe marker gene mutation, thus is starved for the protein activity andrepeatedly signals the production of the protein.

As used herein, “gene deletion” refers to an amount of DNA copy numberless than 2 and “amplification” refers to an amount of DNA copy numbergreater than 2. A “diploid” amount refers to a copy number equal to 2.The term “diploid or amplification” can be interpreted as “not deletion”of a gene copy. In a marker whose alternative informative amount is genedeletion, amplification generally would not be seen. Conversely, theterm “diploid or deletion” can be interpreted as “not amplification” ofcopy number. In a marker whose alternative informative amount isamplification, gene deletion generally would not be seen. For the sakeof clarity, sequence deletion can occur within a gene as a result ofmarker gene mutation and can result in absence of transcribed protein ora shortened mRNA or protein. Such a deletion may not affect copy number.

The terms “long term survival” and “short term survival” refer to thelength of time after receiving a first dose of treatment that a cancerpatient is predicted to live. A “long term survivor” refers to a patientexpected have a slower rate of progression or later death from the tumorthan those patients identified as short term survivors. “Enhancedsurvival” or “a slower rate of death” are estimated life spandeterminations based upon characteristic, e.g., size, sequence,composition or amount of one or more of markers described herein, e.g.,as compared to a reference standard such that 70%, 80%, 90% or more ofthe population will be alive a sufficient time period after receiving afirst dose of treatment. A “faster rate of death” or “shorter survivaltime” refer to estimated life span determinations based uponcharacteristic, e.g., size, sequence, composition or amount of one ormore of markers described herein, e.g., as compared to a referencestandard such that 50%, 40%, 30%, 20%, 10% or less of the populationwill not live a sufficient time period after receiving a first dose oftreatment. In some embodiments, the sufficient time period is at least6, 12, 18, 24 or 30 months measured from the first day of receiving acancer therapy.

A cancer is “responsive” to a therapeutic agent or there is a “goodresponse” to a treatment if its rate of growth is inhibited as a resultof contact with the therapeutic agent, compared to its growth in theabsence of contact with the therapeutic agent. Growth of a cancer can bemeasured in a variety of ways, for instance, the characteristic, e.g.,size of a tumor or the expression of tumor markers appropriate for thattumor type may be measured. For example, the response definitions usedto support the identification of markers associated with myeloma and itsresponse to an NAE inhibitor, such as a 1-substituted methyl sulfamatetherapy, the Southwestern Oncology Group (SWOG) criteria as described inBlade et al. (1998) Br J Haematol. 102:1115-23 can be used. Thesecriteria define the type of response measured in myeloma and also thecharacterization of time to disease progression which is anotherimportant measure of a tumor's sensitivity to a therapeutic agent. Forsolid tumors, the Response Evaluation Criteria in Solid Tumors (RECIST)guidelines (Eisenhauer et al. (2009) E. J. Canc. 45:228-247) can be usedto support the identification of markers associated with solid tumorsand response of solid tumors to an NAE inhibitor. International WorkingGroups convene periodically to set, update and publish response criteriafor various types of cancers. Such published reports can be followed tosupport the identification of markers of the subject tumors and theirresponse to NAE inhibitors. Examples are criteria for Acute MyelogenousLeukemia (AML, Cheson et al. (2003) J. Clin. Oncol. 21:4642-4649),lymphomas, e.g., non-Hodgkin's and Hodgkin's lymphoma (Cheson et al.(2007) J. Clin. Oncol. 25:579-596). Criteria take into account analysismethods such as Positron Emission Tomography (PET), e.g., foridentifying sites with measurable altered metabolic activity (e.g., attumor sites) or to trace specific markers into tumors in vivo,immunohistochemistry, e.g., to identify tumor cells by detecting bindingof antibodies to specific tumor markers, and flow cytometry, e.g., tocharacterize cell types by differential markers and fluorescent stains,in addition to traditional methods such as histology to identify cellcomposition (e.g., blast counts in a blood smear or a bone marrowbiopsy, presence and number of mitotic figures) or tissue structure(e.g., disordered tissue architecture or cell infiltration of basementmembrane). The quality of being responsive to an NAE inhibitor, such asa 1-substituted methyl sulfamate therapy can be a variable one, withdifferent cancers exhibiting different levels of “responsiveness” to agiven therapeutic agent, under different conditions. Still further,measures of responsiveness can be assessed using additional criteriabeyond growth size of a tumor, including patient quality of life, degreeof metastases, etc. In addition, clinical prognostic markers andvariables can be assessed (e.g., M protein in myeloma, PSA levels inprostate cancer) in applicable situations.

A cancer is “non-responsive” or has a “poor response” to a therapeuticagent or there is a poor response to a treatment if its rate of growthis not inhibited, or inhibited to a very low degree, as a result ofcontact with the therapeutic agent when compared to its growth in theabsence of contact with the therapeutic agent. As stated above, growthof a cancer can be measured in a variety of ways, for instance, the sizeof a tumor or the expression of tumor markers appropriate for that tumortype may be measured. For example, the response definitions used tosupport the identification of markers associated with non-response oftumors to therapeutic agents, guidelines such as those described abovecan be used. The quality of being non-responsive to a therapeutic agentcan be a highly variable one, with different cancers exhibitingdifferent levels of “non-responsiveness” to a given therapeutic agent,under different conditions. Still further, measures ofnon-responsiveness can be assessed using additional criteria beyondgrowth size of a tumor, including patient quality of life, degree ofmetastases, etc. In addition, clinical prognostic markers and variablescan be assessed (e.g., M protein in myeloma, PSA levels in prostatecancer) in applicable situations.

As used herein, “long time-to-progression, “long TTP” and “shorttime-to-progression,” “short TTP” refer to the amount of time until whenthe stable disease brought by treatment converts into an active disease.On occasion, a treatment results in stable disease which is neither agood nor a poor response, e.g., MR, the disease merely does not getworse, e.g., become a progressive disease, for a period of time. Thisperiod of time can be at least 4-8 weeks, at least 3-6 months or morethan 6 months.

“Treatment” shall mean the use of a therapy to prevent or inhibitfurther tumor growth, as well as to cause shrinkage of a tumor, and toprovide longer survival times. Treatment is also intended to includeprevention of metastasis of tumor. A tumor is “inhibited” or “treated”if at least one symptom (as determined byresponsiveness/non-responsiveness, time to progression, or indicatorsknown in the art and described herein) of the cancer or tumor isalleviated, terminated, slowed, minimized, or prevented. Anyamelioration of any symptom, physical or otherwise, of a tumor pursuantto treatment using a therapeutic regimen (e.g., NAE inhibitor, such as a1-substituted methyl sulfamate regimen) as further described herein, iswithin the scope of the invention.

As used herein, the term “agent” is defined broadly as anything thatcancer cells, including tumor cells, may be exposed to in a therapeuticprotocol. In the context of the present invention, such agents include,but are not limited to, an NAE inhibitor, such as a 1-substituted methylsulfamate agents, as well as chemotherapeutic agents as known in the artand described in further detail herein.

The term “probe” refers to any molecule, e.g., an isolated molecule,which is capable of selectively binding to a specifically intendedtarget molecule, for example a marker of the invention. Probes can beeither synthesized by one skilled in the art, or derived fromappropriate biological preparations. For purposes of detection of thetarget molecule, probes may be specifically designed to be labeled, asdescribed herein. Examples of molecules that can be utilized as probesinclude, but are not limited to, RNA, DNA, proteins, antibodies, andorganic monomers.

A “normal” characteristic, e.g., size, sequence, composition or amountof a marker may refer to the characteristic, e.g., size, sequence,composition or amount in a “reference sample.” A reference sample can bea matched normal, e.g., germline, sample from the same patient from whomthe tumor, e.g., with a somatic mutation, is derived. A reference samplecan be a sample from a healthy subject not having the marker-associateddisease or a reference characteristic e.g., the average characteristic,e.g., size, sequence, composition or amount of the wild type marker inseveral healthy subjects. A reference sample characteristic, e.g., size,sequence, composition or amount may be comprised of a characteristic,e.g., size, sequence, composition or amount of one or more markers froma reference database. Alternatively, a “normal” characteristic, e.g.,size, sequence, composition or level of expression of a marker is thecharacteristic, e.g., size, sequence, composition or amount of themarker, e.g., marker gene in non-tumor cells in a similar environment orresponse situation from the same patient from whom the tumor is derived.The normal amount of DNA copy number is 2 or diploid, with the exceptionof X-linked genes in males, where the normal DNA copy number is 1.

“Over-expression” and “under-expression” of a marker gene, refer toexpression of the marker gene of a patient at a greater or lesser level(e.g. more than three-halves-fold, at least two-fold, at leastthree-fold, greater or lesser level etc.), respectively, than normallevel of expression of the marker gene, e.g., as measured by mRNA orprotein, in a test sample that is greater than the standard error of theassay employed to assess expression. A “significant” expression levelmay refer to a level which either meets or is above or below apre-determined score for a marker gene set as determined by methodsprovided herein.

“Complementary” refers to the broad concept of sequence complementaritybetween regions of two nucleic acid strands or between two regions ofthe same nucleic acid strand. It is known that an adenine residue of afirst nucleic acid region is capable of forming specific hydrogen bonds(“base pairing”) with a residue of a second nucleic acid region which isantiparallel to the first region if the residue is thymine or uracil.Similarly, it is known that a cytosine residue of a first nucleic acidstrand is capable of base pairing with a residue of a second nucleicacid strand which is antiparallel to the first strand if the residue isguanine. A first region of a nucleic acid is complementary to a secondregion of the same or a different nucleic acid if, when the two regionsare arranged in an antiparallel fashion, at least one nucleotide residueof the first region is capable of base pairing with a residue of thesecond region. In an embodiment, the first region comprises a firstportion and the second region comprises a second portion, whereby, whenthe first and second portions are arranged in an antiparallel fashion,at least about 50%, at least about 75%, at least about 90%, or at leastabout 95% or all of the nucleotide residues of the first portion arecapable of base pairing with nucleotide residues in the second portion.

“Homologous” as used herein, refers to nucleotide sequence similaritybetween two regions of the same nucleic acid strand or between regionsof two different nucleic acid strands. When a nucleotide residueposition in both regions is occupied by the same nucleotide residue,then the regions are homologous at that position. A first region ishomologous to a second region if at least one nucleotide residueposition of each region is occupied by the same residue. Homologybetween two regions is expressed in terms of the proportion ofnucleotide residue positions of the two regions that are occupied by thesame nucleotide residue (i.e., by percent identity). By way of example,a region having the nucleotide sequence 5′-ATTGCC-3′ and a region havingthe nucleotide sequence 5′-TATGGC-3′ share homology with 50% identity.In one embodiment, the first region comprises a first portion and thesecond region comprises a second portion, whereby, at least about 50%,at least about 75%, at least about 90%, or at least about 95% of thenucleotide residue positions of each of the portions are occupied by thesame nucleotide residue. In an embodiment of 100% identity, allnucleotide residue positions of each of the portions are occupied by thesame nucleotide residue.

Unless otherwise specified herewithin, the terms “antibody” and“antibodies” broadly encompass naturally-occurring forms of antibodies,e.g., polyclonal antibodies (e.g., IgG, IgA, IgM, IgE) and monoclonaland recombinant antibodies such as single-chain antibodies, two-chainand multi-chain proteins, chimeric, CDR-grafted, human and humanizedantibodies and multi-specific antibodies, as well as fragments andderivatives of all of the foregoing, which fragments (e.g., dAbs, scFv,Fab, F(ab)′₂, Fab′) and derivatives have at least an antigenic bindingsite. Antibody derivatives may comprise a protein or chemical moietyconjugated to an antibody. The term “antibody” also includes syntheticand genetically engineered variants.

A “kit” is any article of manufacture (e.g., a package or container)comprising at least one reagent, e.g. a probe, for specificallydetecting a marker or marker set of the invention. The article ofmanufacture may be promoted, distributed, sold or offered for sale as aunit for performing, e.g., in vitro, the methods of the presentinvention, e.g., on a sample having been obtained from a patient. Thereagents included in such a kit can comprise at least one nucleic acidprobe and, optionally, one or more primers and/or antibodies for use indetecting marker characteristics, e.g., size, sequence composition oramount, e.g., expression. In addition, a kit of the present inventioncan contain instructions which describe a suitable detection assay. Sucha kit can be conveniently used, e.g., in a clinical or a contracttesting setting, to generate information, e.g., on expression levels,characteristic, e.g., size, sequence or composition of one or moremarker, to be recorded, stored, transmitted or received to allow fordiagnosis, evaluation or treatment of patients exhibiting symptoms ofcancer, in particular patients exhibiting the possible presence of acancer capable of treatment with NAE inhibition therapy, including,e.g., hematological cancers e.g., myelomas (e.g., multiple myeloma),lymphomas (e.g., non-hodgkins lymphoma), leukemias (e.g., acutemyelogenous leukemia), and solid tumors (e.g., tumors of skin, lung,breast, ovary, etc.).

The present methods and compositions are designed for use in diagnosticsand therapeutics for a patient suffering from cancer. A cancer or tumoris treated or diagnosed according to the present methods. “Cancer” or“tumor” is intended to include any neoplastic growth in a patient,including an initial tumor and any metastases. The cancer can be of thehematological or solid tumor type. Hematological tumors include tumorsof hematological origin, including, e.g., myelomas (e.g., multiplemyeloma), leukemias (e.g., Waldenstrom's syndrome, chronic lymphocyticleukemia, acute myelogenous leukemia, chronic myelogenous leukemia,other leukemias), lymphomas (e.g., B-cell lymphomas, non-Hodgkin'slymphoma) and myelodysplastic syndrome. Solid tumors can originate inorgans, and include cancers such as in skin, lung, brain, breast,prostate, ovary, colon, kidney, pancreas, liver, esophagus, stomach,intestine, bladder, uterus, cervix, head and neck, central nervoussystem, bone, testis, adrenal gland, etc. The cancer can comprise a cellin which a marker gene has a mutation. As used herein, cancer cells,including tumor cells, refer to cells that divide at an abnormal(increased) rate or whose control of growth or survival is differentthan for cells in the same tissue where the cancer cell arises or lives.Cancer cells include, but are not limited to, cells in carcinomas, suchas squamous cell carcinoma, basal cell carcinoma, sweat gland carcinoma,sebaceous gland carcinoma, adenocarcinoma, papillary carcinoma,papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma,undifferentiated carcinoma, bronchogenic carcinoma, melanoma, renal cellcarcinoma, hepatoma-liver cell carcinoma, bile duct carcinoma,cholangiocarcinoma, papillary carcinoma, transitional cell carcinoma,choriocarcinoma, semonoma, embryonal carcinoma, mammary carcinomas,gastrointestinal carcinoma, colonic carcinomas, bladder carcinoma,prostate carcinoma, and squamous cell carcinoma of the neck and headregion; sarcomas, such as fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordosarcoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, synoviosarcoma andmesotheliosarcoma; hematologic cancers, such as myelomas, leukemias(e.g., acute myelogenous leukemia, chronic lymphocytic leukemia,granulocytic leukemia, monocytic leukemia, lymphocytic leukemia), andlymphomas (e.g., follicular lymphoma, mantle cell lymphoma, diffuselarge Bcell lymphoma, malignant lymphoma, plasmocytoma, reticulum cellsarcoma, or Hodgkins disease); and tumors of the nervous systemincluding glioma, meningoma, medulloblastoma, schwannoma or epidymoma.

As used herein, the term “noninvasive” refers to a procedure whichinflicts minimal harm to a subject. In the case of clinicalapplications, a noninvasive sampling procedure can be performed quickly,e.g., in a walk-in setting, typically without anaesthesia and/or withoutsurgical implements or suturing. Examples of noninvasive samples includeblood, serum, saliva, urine, buccal swabs, throat cultures, stoolsamples and cervical smears. Noninvasive diagnostic analyses includex-rays, magnetic resonance imaging, positron emission tomography, etc.

Described herein is the assessment of outcome for treatment of a tumorthrough measurement of the amount of pharmacogenomic markers. Alsodescribed are assessing the outcome by noninvasive, convenient orlow-cost means, for example, from blood samples. Typical methods todetermine extent of cancer or outcome of a hematological tumor, e.g.,lymphoma, leukemia, e.g., acute myelogenous leukemia, myeloma (e.g.,multiple myeloma) can employ bone marrow biopsy to collect tissue forgenotype or phenotype, e.g., histological analysis. The inventionprovides methods for determining, assessing, advising or providing anappropriate therapy regimen for treating a tumor or managing disease ina patient. Monitoring a treatment using the kits and methods disclosedherein can identify the potential for unfavorable outcome and allowtheir prevention, and thus a savings in morbidity, mortality andtreatment costs through adjustment in the therapeutic regimen, cessationof therapy or use of alternative therapy.

The term “biological sample” is intended to include a patient sample,e.g., tissue, cells, biological fluids and isolates thereof, isolatedfrom a subject, as well as tissues, cells and fluids present within asubject and can be obtained from a patient or a normal subject. Inhematological tumors of the bone marrow, e.g., myeloma tumors, primaryanalysis of the tumor can be performed on bone marrow samples. However,some tumor cells, (e.g., clonotypic tumor cells, circulating endothelialcells), are a percentage of the cell population in whole blood. Thesecells also can be mobilized into the blood during treatment of thepatient with granulocyte-colony stimulating factor (G-CSF) inpreparation for a bone marrow transplant, a standard treatment forhematological tumors, e.g., leukemias, lymphomas and myelomas. Examplesof circulating tumor cells in multiple myeloma have been studied e.g.,by Filarski et al. (2000) Blood 95:1056-65 and Rigalin et al. (2006)Blood 107:2531-5. Thus, noninvasive samples, e.g., for in vitromeasurement of markers to determine outcome of treatment, can includeperipheral blood samples. Accordingly, cells within peripheral blood canbe tested for marker amount. For patients with hematological tumors, acontrol, reference sample for normal characteristic, e.g., size,sequence, composition or amount can be obtained from skin or a buccalswab of the patient. For solid tumors, a typical tumor sample is abiopsy of the tumor and thus comprises solid tumor cells. Alternatively,a sample of tumor cells shed or scraped from the tumor site can becollected noninvasively, such as in blood, sputum, a nipple aspirate,urine, stool, cervical smear, etc. For solid tumors, a control referencesample for normal characteristic, e.g., size, sequence, composition oramount can be obtained from blood of the patient.

Blood collection containers can comprise an anti-coagulant, e.g.,heparin or ethylene-diaminetetraacetic acid (EDTA), sodium citrate orcitrate solutions with additives to preserve blood integrity, such asdextrose or albumin or buffers, e.g., phosphate. If the amount of markeris being measured by measuring the level of its DNA in the sample, a DNAstabilizer, e.g., an agent that inhibits DNAse, can be added to thesample. If the amount of marker is being measured by measuring the levelof its RNA in the sample, an RNA stabilizer, e.g., an agent thatinhibits RNAse, can be added to the sample. If the amount of marker isbeing measured by measuring the level of its protein in the sample, aprotein stabilizer, e.g., an agent that inhibits proteases, can be addedto the sample. An example of a blood collection container is PAXGENE®tubes (PREANALYTIX, Valencia, Calif.), useful for RNA stabilization uponblood collection. Peripheral blood samples can be modified, e.g.,fractionated, sorted or concentrated (e.g., to result in samplesenriched with tumor or depleted of tumor (e.g., for a referencesample)). Examples of modified samples include clonotypic myeloma cells,which can be collected by e.g., negative selection, e.g., separation ofwhite blood cells from red blood cells (e.g., differentialcentrifugation through a dense sugar or polymer solution (e.g., FICOLL®solution (Amersham Biosciences division of GE healthcare, Piscataway,N.J.) or HISTOPAQUE®-1077 solution, Sigma-Aldrich Biotechnology LP andSigma-Aldrich Co., St. Louis, Mo.)) and/or positive selection by bindingB cells to a selection agent (e.g., a reagent which binds to a tumorcell or myeloid progenitor marker, such as CD34, CD38, CD138, or CD133,for direct isolation (e.g., the application of a magnetic field tosolutions of cells comprising magnetic beads (e.g., from MiltenyiBiotec, Auburn, Calif.) which bind to the B cell markers) orfluorescent-activated cell sorting).

Alternatively, a tumor cell line, e.g., OCI-Ly3, OCI-Ly10 cell (Alizadehet al. (2000) Nature 403:503-511), a RPMI 6666 cell, a SUP-B15 cell, aKG-1 cell, a CCRF-SB cell, an 8ES cell, a Kasumi-1 cell, a Kasumi-3cell, a BDCM cell, an HL-60 cell, a Mo-B cell, a JM1 cell, a GA-10 cellor a B-cell lymphoma (e.g., BC-3) or a cell line or a collection oftumor cell lines (see e.g., McDermott et al. (2007) PNAS 104:19936-19941or ONCOPANEL™ anti-cancer tumor cell profiling screen (RicercaBiosciences, Bothell, Wash.)) can be assayed. A skilled artisan readilycan select and obtain the appropriate cells (e.g., from American TypeCulture Collection (ATCC®), Manassas, Va.) that are used in the presentmethod. If the compositions or methods are being used to predict outcomeof treatment in a patient or monitor the effectiveness of a therapeuticprotocol, then a tissue or blood sample having been obtained from thepatient being treated is a useful source of cells or marker gene or geneproducts for an assay.

The sample, e.g., tumor, e.g., biopsy or bone marrow, blood or modifiedblood, (e.g., comprising tumor cells) and/or the reference, e.g.,matched control (e.g., germline), sample can be subjected to a varietyof well-known post-collection preparative and storage techniques (e.g.,nucleic acid and/or protein extraction, fixation, storage, freezing,ultrafiltration, concentration, evaporation, centrifugation, etc.) priorto assessing the amount of the marker in the sample.

In an embodiment, mutational status of a marker gene, e.g., a mutationin a marker can be identified by sequencing a nucleic acid, e.g., a DNA,RNA, cDNA or a protein correlated with the marker gene. There areseveral sequencing methods known in the art to sequence nucleic acids. Anucleic acid primer can be designed to bind to a region comprising apotential mutation site or can be designed to complement the mutatedsequence rather than the wild type sequence. Primer pairs can bedesigned to bracket a region comprising a potential mutation in a markergene. A primer or primer pair can be used for sequencing one or bothstrands of DNA corresponding to the marker gene. A primer can be used inconjunction with a probe, e.g., a nucleic acid probe, e.g., ahybridization probe, to amplify a region of interest prior to sequencingto boost sequence amounts for detection of a mutation in a marker gene.Examples of regions which can be sequenced include an entire gene,transcripts of the gene and a fragment of the gene or the transcript,e.g., one or more of exons or untranslated regions. Examples ofmutations to target for primer selection and sequence or compositionanalysis can be found in public databases which collect mutationinformation, such as COSMIC and dbGaP. Some mutations of marker genessuch as NF2, SMAD, KDM6A or FBXW7 are listed in Tables 8-11 in theExamples as examples of mutations that can be associated withsensitivity to NAE inhibition, e.g., inhibition by 1-methyl sulfamates,e.g., MLN4924.

Sequencing methods are known to one skilled in the art. Examples ofmethods include the Sanger method, the SEQUENOM™ method and NextGeneration. Sequencing (NGS) methods. The Sanger method, comprisingusing electrophoresis, e.g., capillary electrophoresis to separateprimer-elongated labeled DNA fragments, can be automated forhigh-throughput applications. The primer extension sequencing can beperformed after PCR amplification of regions of interest. Software canassist with sequence base calling and with mutation identification.SEQUENOM™ MASSARRAY® sequencing analysis (San Diego, Calif.) is amass-spectrometry method which compares actual mass to expected mass ofparticular fragments of interest to identify mutations. NGS technology(also called “massively parallel sequencing” and “second generationsequencing”) in general provides for much higher throughput thanprevious methods and uses a variety of approaches (reviewed in Zhang etal. (2011) J. Genet. Genomics 38:95-109 and Shendure and Hanlee (2008)Nature Biotech. 26:1135-1145). NGS methods can identify low frequencymutations in a marker in a sample. Some NGS methods (see, e.g., GS-FLXGenome Sequencer (Roche Applied Science, Branford, Conn.), Genomeanalyzer (Illumina, Inc. San Diego, Calif.) SOLID™ analyzer (AppliedBiosystems, Carlsbad, Calif.), Polonator G.007 (Dover Systems, Salem,N.H.), HELISCOPE™ (Helicos Biosciences Corp., Cambridge, Mass.)) usecyclic array sequencing, with or without clonal amplification of PCRproducts spatially separated in a flow cell and various schemes todetect the labeled modified nucleotide that is incorporated by thesequencing enzyme (e.g., polymerase or ligase). In one NGS method,primer pairs can be used in PCR reactions to amplify regions ofinterest. Amplified regions can be ligated into a concatenated product.Clonal libraries are generated in the flow cell from the PCR or ligatedproducts and further amplified (“bridge” or “cluster” PCR) forsingle-end sequencing as the polymerase adds a labeled, reversiblyterminated base that is imaged in one of four channels, depending on theidentity of the labeled base and then removed for the next cycle.Software can aid in the comparison to genomic sequences to identifymutations.

Composition of proteins and nucleic acids can be determined by many waysknown in the art, such as by treating them in ways that cleave, degradeor digest them and then analyzing the components. Mass spectrometry,electrophoresis and chromatography can separate and define componentsfor comparison. Mutations which cause deletions or insertions can beidentified by size or charge differences in these methods. Proteindigestion or restriction enzyme nucleic acid digestion can revealdifferent fragment patterns after some mutations. Antibodies thatrecognize particular mutant amino acids in their structural contexts canidentify and detect these mutations in samples (see below).

In an embodiment, DNA, e.g., genomic DNA corresponding to the wild typeor mutated marker can be analyzed both by in situ and by in vitroformats in a biological sample using methods known in the art. DNA canbe directly isolated from the sample or isolated after isolating anothercellular component, e.g., RNA or protein. Kits are available for DNAisolation, e.g., QIAAMP® DNA Micro Kit (Qiagen, Valencia, Calif.). DNAalso can be amplified using such kits.

In another embodiment, mRNA corresponding to the marker can be analyzedboth by in situ and by in vitro formats in a biological sample usingmethods known in the art. An example of a method for measuringexpression level is included in the Examples. For example a nucleic acidprobe can be used to hybridize to a marker and the amount of probehybridized can be measured. Many expression detection methods useisolated RNA. For in vitro methods, any RNA isolation technique thatdoes not select against the isolation of mRNA can be utilized for thepurification of RNA from tumor cells (see, e.g., Ausubel et al., ed.,Current Protocols in Molecular Biology, John Wiley & Sons, New York1987-1999). Additionally, large numbers of tissue samples can readily beprocessed using techniques well known to those of skill in the art, suchas, for example, the single-step RNA isolation process of Chomczynski(1989, U.S. Pat. No. 4,843,155). RNA can be isolated using standardprocedures (see e.g., Chomczynski and Sacchi (1987) Anal. Biochem.162:156-159), solutions (e.g., trizol, TRI REAGENT® (Molecular ResearchCenter, Inc., Cincinnati, Ohio; see U.S. Pat. No. 5,346,994) or kits(e.g., a QIAGEN® Group RNEASY® isolation kit (Valencia, Calif.) orLEUKOLOCK™ Total RNA Isolation System, Ambion division of AppliedBiosystems, Austin, Tex.).

Additional steps may be employed to remove DNA from RNA samples. Celllysis can be accomplished with a nonionic detergent, followed bymicrocentrifugation to remove the nuclei and hence the bulk of thecellular DNA. DNA subsequently can be isolated from the nuclei for DNAanalysis. In one embodiment, RNA is extracted from cells of the varioustypes of interest using guanidinium thiocyanate lysis followed by CsClcentrifugation to separate the RNA from DNA (Chirgwin et al. (1979)Biochemistry 18:5294-99). Poly(A)+RNA is selected by selection witholigo-dT cellulose (see Sambrook et al. (1989) Molecular Cloning—ALaboratory Manual (2nd ed.), Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.). Alternatively, separation of RNA from DNA can beaccomplished by organic extraction, for example, with hot phenol orphenol/chloroform/isoamyl alcohol. If desired, RNAse inhibitors may beadded to the lysis buffer. Likewise, for certain cell types, it may bedesirable to add a protein denaturation/digestion step to the protocol.For many applications, it is desirable to enrich mRNA with respect toother cellular RNAs, such as transfer RNA (tRNA) and ribosomal RNA(rRNA). Most mRNAs contain a poly(A) tail at their 3′ end. This allowsthem to be enriched by affinity chromatography, for example, usingoligo(dT) or poly(U) coupled to a solid support, such as cellulose orSEPHADEX® medium (see Ausubel et al. (1994) Current Protocols InMolecular Biology, vol. 2, Current Protocols Publishing, New York). Oncebound, poly(A)+mRNA is eluted from the affinity column using 2 mMEDTA/0.1% SDS.

The characteristic of a marker of the invention in a biological sample,e.g., after obtaining a biological sample (e.g., a bone marrow sample, atumor biopsy or a reference sample) from a test subject, may be assessedby any of a wide variety of well known methods for detecting ormeasuring the characteristic, e.g., of a nucleic acid (e.g., RNA, mRNA,genomic DNA, or cDNA) and/or translated protein. Non-limiting examplesof such methods include immunological methods for detection of secreted,cell-surface, cytoplasmic, or nuclear proteins, protein purificationmethods, protein function or activity assays, nucleic acid hybridizationmethods, nucleic acid reverse transcription methods, and nucleic acidamplification methods. These methods include gene array/chip technology,RT-PCR, TAQMAN® gene expression assays (Applied Biosystems, Foster City,Calif.), e.g., under GLP approved laboratory conditions, in situhybridization, immunohistochemistry, immunoblotting, FISH (flourescencein situ hybridization), FACS analyses, northern blot, southern blot,INFINIUM® DNA analysis Bead Chips (Illumina, Inc., San Diego, Calif.),quantitative PCR, bacterial artificial chromosome arrays, singlenucleotide polymorphism (SNP) arrays (Affymetrix, Santa Clara, Calif.)or cytogenetic analyses. The detection methods of the invention can thusbe used to detect RNA, mRNA, protein, cDNA, or genomic DNA, for example,in a biological sample in vitro as well as in vivo. Furthermore, in vivotechniques for detection of a polypeptide or nucleic acid correspondingto a marker of the invention include introducing into a subject alabeled probe to detect the biomarker, e.g., a nucleic acidcomplementary to the transcript of a biomarker or a labeled antibody, Fcreceptor or antigen directed against the polypeptide, e.g., wild type ormutant marker. For example, the antibody can be labeled with aradioactive isotope whose presence and location in a subject can bedetected by standard imaging techniques. These assays can be conductedin a variety of ways. A skilled artisan can select from these or otherappropriate and available methods based on the nature of the marker(s),tissue sample and mutation in question. Some methods are described inmore detail in later sections. Different methods or combinations ofmethods could be appropriate in different cases or, for instance indifferent types of tumors or patient populations.

In vitro techniques for detection of a polypeptide corresponding to amarker of the invention include enzyme linked immunosorbent assays(ELISAs), Western blots, protein array, immunoprecipitation andimmunofluorescence. In such examples, expression of a marker is assessedusing an antibody (e.g., a radio-labeled, chromophore-labeled,fluorophore-labeled, or enzyme-labeled antibody), an antibody derivative(e.g., an antibody conjugated with a substrate or with the protein orligand of a protein-ligand pair (e.g., biotin-streptavidin)), or anantibody fragment (e.g., a single-chain antibody, an isolated antibodyhypervariable domain, etc.) which binds specifically with a markerprotein or fragment thereof, e.g., a protein or fragment comprising aregion which can be mutated or a portion comprising a mutated sequence,or a mutated residue in its structural context, including a markerprotein which has undergone all or a portion of its normalpost-translational modification. An antibody can detect a protein withan amino acid sequence selected from the group consisting of SEQ IDNO:3, 6, 10, 11, 14, 17, 20 and 23. Alternatively, an antibody candetect a mutated protein with a variant amino acid sequence selectedfrom the group consisting of a mutant of SEQ ID NO:3, 6, 10, 11, 14, 17,20 and 23. Residues listed as mutated in public databases such as COSMICof dbGaP can be prepared in immunogenic compositions for generation ofantibodies that will specifically recognize and bind to the mutantresidues. Another method can employ pairs of antibodies, wherein one ofthe pair would bind a marker protein upstream, i.e. N-terminal to theregion of expected mutation, e.g., nonsense or deletion and the other ofthe pair would bind the protein downstream. Wild type protein would bindboth antibodies of the pair, but a protein with a nonsense or deletionmutation would bind only the N-terminal antibody of the pair. An assaysuch as a sandwich ELISA assay could detect a loss of quantity of thewild type protein in the tumor sample, e.g., in comparison to thereference sample, or a standard ELISA would comparison of the levels ofbinding of the antibodies to infer that a mutation is present in a tumorsample.

Indirect methods for determining the amount or functionality of aprotein marker also include measurement of the activity of the protein.For example, a sample, or a protein isolated from the sample orexpressed from nucleic acid isolated, cloned or amplified from thesample can be assessed for marker protein activity. NF2 activity can bemeasured by its ability to associate with binding partners, e.g., in acell-free assay or in a cell-based assay. In an example, the ability ofNF2 to bind to red blood cell membranes or p55/MPP1 can be measured (Seoet al. (2009) Exp. Biol. Med. 234:255-262). In another example, SMAD4activity can be measured by its activity in signal transduction, e.g.,in a cell-free assay or in a cell-based assay. In an example, thephosphorylation state of SMAD4 can be measured, the binding of SMAD4 toDNA at a Smad-binding element, e.g., in a gel shift assay or in areporter assay (see, e.g., Kuang and Chen (2004) Oncogene 23:1021-1029),can be measured or the translocation of SMAD4 between the nucleus andcytoplasm can be visualized and quantified on cell images. In anotherexample KDM6A activity can be measured by its ability to demethylateproteins, e.g., histones. For example, an assay can measure the level ofdemethylation of lysine 27 of histone 3 (Hong et al. (2007) PNAS104:18439-18444). In another example, FBXW7 activity can be measured byits ability to bind cyclin E or to associate into the Skp-cullin-F-boxubiquitin ligase complex. In another example, TP53 activity can bemeasured by the ability to bind to DNA or to form tetramers.

In one embodiment, expression of a marker is assessed by preparingmRNA/cDNA (i.e., a transcribed polynucleotide) from cells in a patientsample, and by hybridizing the mRNA/cDNA with a referencepolynucleotide, e.g., an isolated nucleic acid probe, e.g., ahybridization probe, which is a complement of a marker nucleic acid, ora fragment thereof. cDNA can, optionally, be amplified using any of avariety of polymerase chain reaction methods prior to hybridization withthe reference polynucleotide. Expression of one or more markers likewisecan be detected using quantitative PCR to assess the level of expressionof the marker(s). An example of the use of measuring mRNA levels is thatan inactivating mutation in a marker gene can result in an altered levelof mRNA in a cell. The level can be upregulated due to feedbacksignaling protein production in view of nonfunctional or absent proteinor downregulated due to instability of an altered mRNA sequence.Alternatively, any of the many known methods of detecting mutations orvariants (e.g. single nucleotide polymorphisms, deletions, etc.,discussed above) of a marker of the invention may be used to detectoccurrence of a mutation in a marker gene in a patient.

An example of direct measurement is quantification of transcripts. Asused herein, the level or amount of expression refers to the absoluteamount of expression of an mRNA encoded by the marker or the absoluteamount of expression of the protein encoded by the marker. As analternative to making determinations based on the absolute expressionamount of selected markers, determinations may be based on normalizedexpression amounts. Expression amount can be normalized by correctingthe absolute expression level of a marker upon comparing its expressionto the expression of a control marker that is not a marker, e.g., in ahousekeeping role that is constitutively expressed. Suitable markers fornormalization also include housekeeping genes, such as the actin gene orbeta-2 microglobulin. Reference markers for data normalization purposesinclude markers which are ubiquitously expressed and/or whose expressionis not regulated by oncogenes. Constitutively expressed genes are knownin the art and can be identified and selected according to the relevanttissue and/or situation of the patient and the analysis methods. Suchnormalization allows one to compare the expression level in one sample,to another sample, e.g., between samples from different times ordifferent subjects. Further, the expression level can be provided as arelative expression level. The baseline of a genomic DNA sample, e.g.,diploid copy number, can be determined by measuring amounts in cellsfrom subjects without a tumor or in non-tumor cells from the patient. Todetermine a relative amount of a marker or marker set, the amount of themarker or marker set is determined for at least 1, or 2, 3, 4, 5, ormore samples, e.g., 7, 10, 15, 20 or 50 or more samples in order toestablish a baseline, prior to the determination of the expression levelfor the sample in question. To establish a baseline measurement, themean amount or level of each of the markers or marker sets assayed inthe larger number of samples is determined and this is used as abaseline expression level for the biomarkers or biomarker sets inquestion. The amount of the marker or marker set determined for the testsample (e.g., absolute level of expression) is then divided by thebaseline value obtained for that marker or marker set. This provides arelative amount and aids in identifying abnormal levels of markerprotein activity.

Probes based on the sequence of a nucleic acid molecule of the inventioncan be used to detect transcripts or genomic sequences corresponding toone or more markers of the invention. The probe can comprise a labelgroup attached thereto, e.g., a radioisotope, a fluorescent compound, anenzyme, or an enzyme co-factor. Such probes can be used as part of adiagnostic test kit for identifying cells or tissues which express theprotein, such as by measuring levels of a nucleic acid molecule encodingthe protein in a sample of cells from a subject, e.g., detecting mRNAlevels or determining whether a gene encoding the protein has beenmutated or deleted.

In addition to the nucleotide sequences described in the databaserecords described herein, it will be appreciated by those skilled in theart that DNA sequence polymorphisms that lead to changes in the aminoacid sequence can exist within a population (e.g., the humanpopulation). Such genetic polymorphisms can exist among individualswithin a population due to naturally occurring allelic variation. Anallele is one of a group of genes which occur alternatively at a givengenetic locus. In addition, it will be appreciated that DNApolymorphisms that affect RNA expression levels can also exist that mayaffect the overall expression level of that gene (e.g., by affectingregulation or degradation).

Primers or nucleic acid probes comprise a nucleotide sequencecomplementary to a specific a marker or a mutated region thereof and areof sufficient length to selectively hybridize with a marker gene ornucleic acid associated with a marker gene, e.g., they can bind to thenucleic acid with base sequence specificity and remain bound, afterwashing. Primers and probes can be used to aid in the isolation andsequencing of marker nucleic acids. In one embodiment, the primer ornucleic acid probe, e.g., a substantially purified oligonucleotide, anisolated nucleic acid, comprises a region having a nucleotide sequencewhich hybridizes, e.g., under stringent conditions, to about 6, 8, 10,12, 15, 20, 25, 30, 40, 50, 60, 75, 100, 200, 350, 500 or moreconsecutive nucleotides of a marker gene or a region comprising amutation in a marker gene or transcript therefrom or a complement. Inanother embodiment, the primer or nucleic acid probe is capable ofhybridizing to a marker nucleic acid comprising a nucleotide sequence ofany sequence set forth in any of SEQ ID NOs:1, 2, 4, 5, 7, 8, 9, 12, 13,15, 16, 18, 19, 21, 22, 24, 25 or a sequence on chromosome 22q from basepair 29999545 to 30094589, chromosome 18q from base pair 48556583 to48611412, chromosome Xp from base pair 44732423 to 44971847, chromosome4q from base pair 153242410 to Ser. No. 15/345,6172, chromosome 17p frombase pair 7571720 to 7590868, chromosome 9p from base pair 21967751 to21994490, or a complement of any of the foregoing. For example, a primeror nucleic acid probe comprising a nucleotide sequence of at least about10 consecutive nucleotides, at least about 15 consecutive nucleotides,at least about 25 consecutive nucleotides, at least about 35 consecutivenucleotides, at least about 50 consecutive nucleotides, or having fromabout 15 to about 20 nucleotides set forth in any of SEQ ID NOs: 1, 2,4, 5, 7, 8, 9, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25 or a sequence onchromosome 22q from base pair 29999545 to 30094589, chromosome 18q frombase pair 48556583 to 48611412, chromosome Xp from base pair 44732423 to44971847, chromosome 4q from base pair 153242410 to Ser. No.15/345,6172, chromosome 17p from base pair 7571720 to 7590868,chromosome 9p from base pair 21967751 to 21994490, or a complement ofany of the foregoing are provided by the invention. Primers or nucleicacid probes having a sequence of more than about 25, 40 or 50nucleotides are also within the scope of the invention. In anotherembodiment, a primer or nucleic acid probe can have a sequence at least70%, at least 75%, 80% or 85%, or at least, 90%, 95% or 97% identical tothe nucleotide sequence of any sequence set forth in any of SEQ ID NOs:1, 2, 4, 5, 7, 8, 9, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25 or asequence on chromosome 22q from base pair 29999545 to 30094589,chromosome 18q from base pair 48556583 to 48611412, chromosome Xp frombase pair 44732423 to 44971847, chromosome 4q from base pair 153242410to Ser. No. 15/345,6172, chromosome 17p from base pair 7571720 to7590868, chromosome 9p from base pair 21967751 to 21994490, or acomplement of any of the foregoing. Nucleic acid analogs can be used asbinding sites for hybridization. An example of a suitable nucleic acidanalogue is peptide nucleic acid (see, e.g., Egholm et al., Nature363:566 568 (1993); U.S. Pat. No. 5,539,083).

In some embodiments, nucleic acid probe can be designed to bind to thewild type sequence, so the presence of a mutation in that region cancause a decrease, e.g., measurable decrease, in binding or hybridizationby that probe. In another embodiment, a nucleic acid probe can bedesigned to bind to a mutant sequence, so the presence of a mutation inthat region can cause an increase in binding or hybridization by thatprobe. In other embodiments, a probe and primer set or a primer pair canbe designed to bracket a region in a marker that can have a mutation soamplification based on that set or pair can result in nucleic acidswhich can be sequenced to identify the mutation.

Primers or nucleic acid probes can be selected using an algorithm thattakes into account binding energies, base composition, sequencecomplexity, cross-hybridization binding energies, and secondarystructure (see Friend et al., International Patent Publication WO01/05935, published Jan. 25, 2001; Hughes et al., Nat. Biotech. 19:342-7(2001). Useful primers or nucleic acid probes of the invention bindsequences which are unique for each transcript, e.g., target mutatedregions and can be used in PCR for amplifying, detecting and sequencingonly that particular nucleic acid, e.g., transcript or mutatedtranscript. Examples of some mutations of marker genes, e.g., NF2,SMAD4, KDM6A and FBXW7 are found in Tables in the Examples (Tables8-11). Other mutations are described in reference articles cited hereinand in public databases described herein. One of skill in the art candesign primers and nucleic acid probes for the markers disclosed hereinor related markers with similar characteristics, e.g., markers on thechromosome loci, or mutations in different regions of the same markergene described herein, using the skill in the art, e.g., adjusting thepotential for primer or nucleic acid probe binding to standardsequences, mutants or allelic variants by manipulating degeneracy or GCcontent in the primer or nucleic acid probe. Computer programs that arewell known in the art are useful in the design of primers with therequired specificity and optimal amplification properties, such as Oligoversion 5.0 (National Biosciences, Plymouth, Minn.). While perfectlycomplementary nucleic acid probes and primers can be used for detectingthe markers described herein and mutants, polymorphisms or allelesthereof, departures from complete complementarity are contemplated wheresuch departures do not prevent the molecule from specificallyhybridizing to the target region. For example, an oligonucleotide primermay have a non-complementary fragment at its 5′ end, with the remainderof the primer being complementary to the target region. Alternatively,non-complementary nucleotides may be interspersed into the nucleic acidprobe or primer as long as the resulting probe or primer is stillcapable of specifically hybridizing to the target region.

An indication of treatment outcome can be assessed by studying theamount of 1 marker, 2 markers, 3 markers or 4 markers, or more, e.g., 5,6, 7, 8, 9, 10, 15, 20, or 25 markers, or mutated portions thereof e.g.,marker genes which participate in or interact with the cullin ringligase pathway e.g., tumor suppressors, e.g., which can be inactivatedby somatic mutation in cancer. Markers can be studied in combinationwith another measure of treatment outcome, e.g., biochemical markers(e.g., M protein in myeloma, kidney health marker such as proteinuria,serum levels of C-reactive protein or cytokeratin 19, cytokeratinfragment 21-1 (CYFRA21-1) for NSCLC, urine levels offibrinogen/fibrinogen degradation products for bladder cancer, urine orblood levels of catecholamines for neuroblastoma, serum levels ofcarbohydrate antigen 19-9 (CA 19-9) or metabolic profiling forpancreatic cancer or blood levels of soluble mesothelin-related peptides(SMRP) in mesothelioma) or histology assessment (e.g., blast count,number of mitotic figures per unit area, depth measurement of invasionof melanoma tumors, esophageal tumors or bladder tumors).

Statistical methods can assist in the determination of treatment outcomeupon measurement of the amount of markers, e.g., measurement of DNA, RNAor protein. The amount of one marker can be measured at multipletimepoints, e.g., before treatment, during treatment, after treatmentwith an agent, e.g., an NAE inhibitor. To determine the progression ofchange in expression of a marker from a baseline, e.g., over time, theexpression results can be analyzed by a repeated measures linearregression model (Littell, Miliken, Stroup, Wolfinger, Schabenberger(2006) SAS for Mixed Models, 2^(nd) edition. SAS Institute, Inc., Cary,N.C.)):

Y _(ijk) −Y _(ij0) =Y _(ij0)+treatment_(i) +day_(k)+(treatment*day)_(ik)+ε_(ijk)  Equation 1

where Y_(ijk) is the log₂ transformed expression (normalized to thehousekeeping genes) on the k^(th) day of the j^(th) animal in the i^(th)treatment, Y_(ij0) is the defined baseline log₂ transformed expression(normalized to the housekeeping genes) of the j^(th) animal in thei^(th) treatment, day_(k) is treated as a categorical variable, andε_(ijk) is the residual error term. A covariance matrix (e.g.,first-order autoregressive, compound symmetry, spatial power law) can bespecified to model the repeated measurements on each animal over time.Furthermore, each treatment time point can be compared back to the sametime point in the vehicle group to test whether the treatment value wassignificantly different from vehicle.

A number of other methods can be used to analyze the data. For instance,the relative expression values could be analyzed instead of the cyclenumber. These values could be examined as either a fold change or as anabsolute difference from baseline. Additionally, a repeated-measuresanalysis of variance (ANOVA) could be used if the variances are equalacross all groups and time points. The observed change from baseline atthe last (or other) time point could be analyzed using a paired t-test,a Fisher exact test (p-value=ΣP(X=x) from x=1 to the number ofsituations, e.g., mutations, tested that show sensitivity to NAEinhibition) for testing significance of data of small sample sizes, or aWilcoxon signed rank test if the data is not normally distributed, tocompare whether a tumor patient was significantly different from anormal subject.

A difference in amount from one timepoint to the next or from the tumorsample to the normal sample can indicate prognosis of treatment outcome.A baseline level can be determined by measuring expression at 1, 2, 3,4, or more times prior to treatment, e.g., at time zero, one day, threedays, one week and/or two weeks or more before treatment. Alternatively,a baseline level can be determined from a number of subjects, e.g.,normal subjects or patients with the same health status or disorder, whodo not undergo or have not yet undergone the treatment, as discussedabove. Alternatively, one can use expression values deposited with theGene Expression Omnibus (GEO) program at the National Center forBiotechnology Information (NCBI, Bethesda, Md.). For example, datasetsof myeloma mRNA expression amounts sampled prior to proteasomeinhibition therapy include GEO Accession number GSE9782, also analyzedin Mulligan, et al. (2006) Blood 109:3177-88 and GSE6477, also analyzedby Cling et al. (2007) Cancer Res. 67:292-9. To test the effect of thetreatment on the tumor, the expression of the marker can be measured atany time or multiple times after some treatment, e.g., after 1 day, 2days, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2months, 3 months and/or 6 or more months of treatment. For example, theamount of a marker can be measured once after some treatment, or atmultiple intervals, e.g., 1-week, 2-week, 4-week or 2-month, 3-month orlonger intervals during treatment. In some embodiments, the measurementof a marker during treatment can be compared to the same markermeasurement at baseline. In other embodiments, the measurement of amarker during treatment can be compared to the same marker measurementat an earlier timepoint. Conversely, to determine onset of progressivedisease after stopping the administration of a therapeutic regimen, theamount of the marker can be measured at any time or multiple timesafter, e.g., 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4weeks, 1 month, 2 months, 3 months and/or 6 or more months after thelast treatment. The measurement of a marker after treatment can becompared to the same marker measurement at the end of treatment. One ofskill in the art would determine the timepoint or timepoints to assessthe amount of the marker depending on various factors, e.g., thepharmacokinetics of the treatment, the treatment duration,pharmacodynamics of the treatment, age of the patient, the nature of thedisorder or mechanism of action of the treatment. A trend in thenegative direction or a decrease in the amount relative to baseline or apre-determined standard of expression of a marker of sensitivity to NAEinhibition therapy, e.g., a decrease in a sensitivity marker identifiedin Table 3, can indicate a decrease in response of the tumor to thetherapy, e.g., increase in resistance. A trend toward a favorableoutcome relative to the baseline or a pre-determined standard ofexpression of a marker of treatment outcome indicates usefulness of thetherapeutic regimen or continued benefit of the therapy. A trend towardan increase in a resistance marker e.g., an increase in a resistancemarker identified in Table 3, can indicate an unfavorable outcome.

Any marker, e.g., marker gene or combination of marker, e.g., markergenes of the invention, or mutations thereof as well as any knownmarkers in combination with the markers, e.g., marker genes of theinvention, may be used in the compositions, kits, and methods of thepresent invention. In general, markers are selected for as great aspossible ability to judge mutational status of a marker gene to predictoutcome of treatment with NAE inhibitor. For example, the choice ofmarkers are selected for as great as possible difference between thecharacteristic, e.g., size, sequence, composition or amount of themarker in samples comprising tumor cells and the characteristic, e.g.,size, sequence, composition or amount of the same marker in controlcells. Although this difference can be as small as the limit ofdetection of the method for assessing the amount of the marker, inanother embodiment, the difference can be at least greater than thestandard error of the assessment method. In the case of RNA or proteinamount, a difference can be at least 1.5-, 2-, 3-, 4-, 5-, 6-, 7-, 8-,9-, 10-, 15-, 20-, 25-, 100-, 500-, 1000-fold or greater. “Low” RNA orprotein amount can be that expression relative to the overall meanacross tumor samples (e.g., hematological tumor, e.g., myeloma) is low.In the case of amount of DNA, e.g., copy number, the amount is 0, 1, 2,3, 4, 5, 6, or more copies. A deletion causes the copy number to be 0 or1; an amplification causes the copy number to be greater than 2. Thedifference can be qualified by a confidence level, e.g., p<0.05, p<0.02,p<0.01 or lower p-value.

Measurement of more than one marker, e.g., a set of 2, 3, 4, 5, 6, 7, 8,9, 10, 12, 15, 20, or 25 or more markers can provide an expressionprofile or a trend indicative of treatment outcome. In some embodiments,the marker set comprises no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 12,15, 20, or 25 markers. In some embodiments, the marker set includes aplurality of chromosome loci, a plurality of marker genes, or aplurality of markers of one or more marker genes (e.g., nucleic acid andprotein, genomic DNA and mRNA, or various combinations of markersdescribed herein). Analysis of treatment outcome through assessing theamount of markers in a set can be accompanied by a statistical method,e.g., a weighted voting analysis which accounts for variables which canaffect the contribution of the amount of a marker in the set to theclass or trend of treatment outcome, e.g., the signal-to-noise ratio ofthe measurement or hybridization efficiency for each marker. A markerset, e.g., a set of 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, or 25 ormore markers, can comprise a primer, probe or primers to analyze atleast one marker DNA or RNA described herein, e.g., a marker onchromosome 22q from base pair 29999545 to 30094589, chromosome 18q frombase pair 48556583 to 48611412, chromosome Xp from base pair 44732423 to44971847, chromosome 4q from base pair 153242410 to Ser. No.15/345,6172, chromosome 17p from base pair 7571720 to 7590868,chromosome 9p from base pair 21967751 to 21994490, NF2, SMAD4, KDM6A,FBXW7, TP53, CDKN2A, CDKN2A_p14, or a complement of any of theforegoing. A marker set, e.g., a set of at least 2, 3, 4, 5, 6, 7, 8, 9,10, 12, 15, 20, or 25 or more markers, can comprise a primer, probe orprimers to detect at least one or at least two or more markers, or atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, or 25 or more mutations onthe markers e.g., of NF2, SMAD4, KDM6A, TP53, CDKN2A, CDKN2A_p14 and/orFBXW7. In another embodiment, a marker set can comprise markers forassessing characteristics of NF2, SMAD4 and/or KDM6A. In an embodiment,a marker set for cancer of the uterus or cervix comprises at least onemarker for assessing at least one characteristic of FBXW7. In anembodiment, a marker set for cancer of the intestine, breast, lung, headand neck, cervix or skin comprises at least one marker for assessing atleast one characteristic of TP53. In an embodiment, a marker set forcancer of the intestine comprises markers for assessing at least onecharacteristic of each of TP53 and APC. In an embodiment, a marker setfor cancer of the skin or central nervous system comprises at least onemarker for assessing at least one characteristic of CDKN2A_p14. In anembodiment, a marker set for cancer of the head and neck or skincomprises at least one marker for assessing at least one characteristicof CDKN2A. In an embodiment, a marker set for cancer of the head andneck comprises at least one marker for assessing at least onecharacteristic of SMAD4. Selected marker sets can be assembled from themarkers provided herein or selected from among markers using methodsprovided herein and analogous methods known in the art. A way to qualifya new marker for use in an assay of the invention is to correlate DNAcopy number in a sample comprising tumor cells with differences inexpression (e.g., fold-change from baseline) of a marker, e.g., a markergene. A useful way to judge the relationship is to calculate thecoefficient of determination r2, after solving for r, the Pearsonproduct moment correlation coefficient and/or preparing a least squaresplot, using standard statistical methods. A correlation can analyze DNAcopy number versus the level of expression of marker, e.g., a markergene. A gene product can be selected as a marker if the result of thecorrelation (r2, e.g., the linear slope of the data in this analysis),is at least 0.1-0.2, at least 0.3-0.5, or at least 0.6-0.8 or more.Markers can vary with a positive correlation to response, TTP orsurvival (i.e., change expression levels in the same manner as copynumber, e.g., decrease when copy number is decreased). Markers whichvary with a negative correlation to copy number (i.e., change expressionlevels in the opposite manner as copy number levels, e.g., increase whencopy number is decreased) provide inconsistent determination of outcome.

Another way to qualify a new marker for use in the assay would be toassay the expression of large numbers of markers in a number of subjectsbefore and after treatment with a test agent. The expression resultsallow identification of the markers which show large changes in a givendirection after treatment relative to the pre-treatment samples. One canbuild a repeated-measures linear regression model to identify the genesthat show statistically significant changes or differences. To then rankthese significant genes, one can calculate the area under the changefrom e.g., baseline vs time curve. This can result in a list of genesthat would show the largest statistically significant changes. Thenseveral markers can be combined together in a set by using such methodsas principle component analysis, clustering methods (e.g., k-means,hierarchical), multivariate analysis of variance (MANOVA), or linearregression techniques. To use such a gene (or group of genes) as amarker, genes which show 2-, 2.5-, 3-, 3.5-, 4-, 4.5-, 5-, 7-, 10-fold,or more differences of expression from baseline would be included in themarker set. An expression profile, e.g., a composite of the expressionlevel differences from baseline or reference of the aggregate marker setwould indicate at trend, e.g., if a majority of markers show aparticular result, e.g., a significant difference from baseline orreference, e.g., 60%, 70%, 80%, 90%, 95% or more markers; or moremarkers, e.g., 10% more, 20% more, 30% more, 40% more, show asignificant result in one direction than the other direction.

In embodiments when the compositions, kits, and methods of the inventionare used for characterizing treatment outcome in a patient, the markeror set of markers of the invention is selected such that a significantresult is obtained in at least about 20%, at least about 40%, 60%, or80%, or in substantially all patients treated with the test agent. Themarker or set of markers of the invention can be selected such that apositive predictive value (PPV) of greater than about 10% is obtainedfor the general population and additional confidence in a marker can beinferred when the PPV is coupled with an assay specificity greater than80%.

Therapeutic Agents

The markers and marker sets of the present invention assess thelikelihood of favorable outcome of therapy (e.g., sensitivity to atherapeutic agent) in patients, e.g., cancer patients, e.g., patientshaving a hematological cancer (e.g., multiple myeloma, leukemias,lymphoma, etc) or solid tumor cancer (e.g., skin cancer such asmelanoma, head and neck cancer, such as esophageal cancer, bladdercancer, lung cancer, such as non-small cell lung cancer (NSCLC),adenocarcinoma of the lung, central nervous system cancer such as lungmetastases in the brain or neuroblastoma, pancreatic cancer, breastcancer, mesothelioma, cervical cancer or intestinal cancer such as colonor rectum adenocarcinoma), based on its ability to affect thecharacteristic, e.g., composition or amount of a marker or markers ofthe invention. Using this prediction, cancer therapies can be evaluatedto design a therapy regimen best suitable for patients in eithercategory.

In particular, the methods can be used to predict patient sensitivity toNAE inhibitors as described in earlier sections. The agents tested inthe present methods can be a single agent or a combination of agents.The methods of the invention include combination of NAE inhibitiontherapy with proteasome inhibition therapy and/or other or additionalagents, e.g., selected from the group consisting of chemotherapeuticagents. For example, the present methods can be used to determinewhether a single chemotherapeutic agent, such as an NAE inhibitor (e.g.,MLN4924) can be used to treat a cancer or whether a one or more agentsshould be used in combination with the NAE inhibitor (e.g., MLN4924).Useful combinations can include agents that have different mechanisms ofaction, e.g., the use of an anti-mitotic agent in combination with analkylating agent and an NAE inhibitor.

As used herein, the term “proteasome inhibitor” refers to any substancewhich directly inhibits enzymatic activity of the 20S or 26S proteasomein vitro or in vivo. In some embodiments, the proteasome inhibitor is apeptidyl boronic acid. Examples of peptidyl boronic acid proteasomeinhibitors suitable for use in the methods of the invention aredisclosed in Adams et al., U.S. Pat. No. 5,780,454 (1998), U.S. Pat. No.6,066,730 (2000), U.S. Pat. No. 6,083,903 (2000); U.S. Pat. No.6,297,217 (2001), U.S. Pat. No. 6,465,433 (2002), U.S. Pat. No.6,548,668 (2003), U.S. Pat. No. 6,617,317 (2003), and U.S. Pat. No.6,747,150 (2004), each of which is hereby incorporated by reference inits entirety, including all compounds and formulae disclosed therein. Insome embodiments, the peptidyl boronic acid proteasome inhibitor isselected from the group consisting of:N(4morpholine)carbonyl-β-(1-naphthyl)-L-alanine-L-leucine boronic acid;N(8quinoline)sulfonyl-β-(1-naphthyl)-L-alanine-L-alanine-L-leucineboronic acid; N(pyrazine)carbonyl-L-phenylalanine-L-leucine boronicacid, and N(4morpholine)-carbonyl-[O-(2-pyridylmethyl)]-L-tyrosine-L-leucine boronicacid. In a particular embodiment, the proteasome inhibitor is N(pyrazine)carbonyl-L-phenylalanine-L-leucine boronic acid (bortezomib;VELCADE®; formerly known as MLN341 or PS-341). Publications describe theuse of the disclosed boronic ester and boronic acid compounds to reducethe rate of muscle protein degradation, to reduce the activity of NF-kBin a cell, to reduce the rate of degradation of p53 protein in a cell,to inhibit cyclin degradation in a cell, to inhibit the growth of acancer cell, and to inhibit NF-kB dependent cell adhesion. Bortezomibspecifically and selectively inhibits the proteasome by binding tightly(Ki=0.6 nM) to one of the enzyme's active sites. Bortezomib isselectively cytotoxic, and has a novel pattern of cytotoxicity inNational Cancer Institute (NCI) in vitro and in vivo assays. Adams J, etal. Cancer Res 59:2615-22. (1999).

Additionally, proteasome inhibitors include peptide aldehyde proteasomeinhibitors (Stein et al., U.S. Pat. No. 5,693,617 (1997); Siman et al.,international patent publication WO 91/13904; Iqbal et al., J. Med.Chem. 38:2276-2277 (1995); and Iinuma et al., international patentpublication WO 05/105826, each of which is hereby incorporated byreference in its entirety), peptidyl epoxy ketone proteasome inhibitors(Crews et al., U.S. Pat. No. 6,831,099; Smyth et al., internationalpatent publication WO 05/111008; Bennett et al., international patentpublication WO 06/045066; Spaltenstein et al. Tetrahedron Lett. 37:1343(1996); Meng, Proc. Natl. Acad. Sci. 96: 10403 (1999); and Meng, CancerRes. 59: 2798 (1999)), alpha-ketoamide proteasome inhibitors (Chatterjeeand Mallamo, U.S. Pat. No. 6,310,057 (2001) and U.S. Pat. No. 6,096,778(2000); and Wang et al., U.S. Pat. No. 6,075,150 (2000) and U.S. Pat.No. 6,781,000 (2004)), peptidyl vinyl ester proteasome inhibitors(Marastoni et al., J. Med. Chem. 48:5038 (2005), and peptidyl vinylsulfone and 2-keto-1,3,4-oxadiazole proteasome inhibitors, such as thosedisclosed in Rydzewski et al., J. Med. Chem. 49:2953 (2006); and Bogyoet aL, Proc. Natl. Acad. Sci. 94:6629 (1997)), azapeptoids and (Bougetet al., Bioorg. Med. Chem. 11:4881 (2003); Baudy-Floc'h et al.,international patent publication WO 05/030707; and Bonnemains et al.,international patent publication WO 03/018557), efrapeptin oligopeptides(Papathanassiu, international patent publication WO 05/115431),lactacystin and salinosporamide and analogs thereof (Fenteany et al.,U.S. Pat. No. 5,756,764 (1998), U.S. Pat. No. 6,147,223 (2000), U.S.Pat. No. 6,335,358 (2002), and U.S. Pat. No. 6,645,999 (2003); Fenteanyet al., Proc. Natl. Acad. Sci. USA (1994) 91:3358; Fenical et al.,international patent publication WO 05/003137; Palladino et al.,international patent publication WO 05/002572; Stadler et al.,international patent publication WO 04/071382; Xiao and Patel, U.S.patent publication 2005/023162; and Corey, international patentpublication WO 05/099687).

Additional therapeutic agents for use in combination with an NAEinhibitor (e.g., MLN4924) in the methods of the invention can comprise aknown class of therapeutic agents comprising glucocorticoid steroids.Glucocorticoid therapy generally comprises at least one glucocorticoidagent (e.g., dexamethasone). In certain applications of the invention,the agent used in methods of the invention is a glucocorticoid agent.One example of a glucocorticoid utilized in the treatment of multiplemyeloma patients as well as other cancer therapies is dexamethasone.Additional glucocorticoids utilized in treatment of hematological andcombination therapy in solid tumors include hydrocortisone, predisolone,prednisone, and triamcinolone.

Other therapeutic agents for use in combination with NAE inhibitiontherapy include chemotherapeutic agents. A “chemotherapeutic agent” isintended to include chemical reagents which inhibit the growth ofproliferating cells or tissues wherein the growth of such cells ortissues is undesirable. Chemotherapeutic agents such as anti-metabolicagents, e.g., Ara AC, 5-FU and methotrexate, antimitotic agents, e.g.,taxane, vinblastine and vincristine, alkylating agents, e.g.,melphanlan, Carmustine (BCNU) and nitrogen mustard, Topoisomerase IIinhibitors, e.g., VW-26, topotecan and Bleomycin, strand-breakingagents, e.g., doxorubicin and Mitoxantrone (DHAD), cross-linking agents,e.g., cisplatin and carboplatin (CBDCA), radiation and ultraviolet lightand are well known in the art (see e.g., Gilman A. G., et al., ThePharmacological Basis of Therapeutics, 8th Ed., Sec 12:1202-1263(1990)), and are typically used to treat neoplastic diseases. Examplesof chemotherapeutic agents generally employed in chemotherapy treatmentsare listed below in Table 2.

TABLE 2 Chemotherapeutic Agents NONPROPRIETARY NAMES CLASS TYPE OF AGENT(OTHER NAMES) Alkylating Nitrogen Mustards Mechlorethamine (HN₂)Cyclophosphamide Ifosfamide Melphalan (L-sarcolysin) ChlorambucilEthylenimines Hexamethylmelamine And Methylmelamines Thiotepa AlkylSulfonates Busulfan Alkylating Nitrosoureas Carmustine (BCNU) Lomustine(CCNU) Semustine (methyl-CCNU) Streptozocin (streptozotocin) AlkylatingTriazenes Decarbazine (DTIC; dimethyltriazenoimi- dazolecarboxamide)Alkylator cis-diamminedichloroplatinum II (CDDP) Antimetabolites FolicAcid Analogs Methotrexate (amethopterin) Pyrimidine Fluorouracil(′5-fluorouracil; 5-FU) Analogs Floxuridine (fluorode-oxyuridine; FUdR)Cytarabine (cytosine arabinoside) Purine Analogs and Mercaptopuine(6-mercaptopurine; 6-MP) Related Thioguanine (6-thioguanine; TG)Inhibitors Pentostatin (2′-deoxycoformycin) Natural Vinca AlkaloidsVinblastin (VLB) Products Vincristine Topoisomerase Etoposide InhibitorsTeniposide Camptothecin Topotecan 9-amino-campotothecin CPT-11 NaturalAntibiotics Dactinomycin (actinomycin D) Products AdriamycinDaunorubicin (daunomycin; rubindomycin) Doxorubicin Bleomycin Plicamycin(mithramycin) Mitomycin (mitomycin C) TAXOL Taxotere EnzymesL-Asparaginase Biological Response Interfon alfa Modifiers Interleukin 2Platinum cis-diamminedichloroplatinum II (CDDP) Coordination CarboplatinComplexes Anthracendione Mitoxantrone Substituted Urea HydroxyureaMiscellaneous Methyl Hydraxzine Procarbazine Agents Derivative(N-methylhydrazine,(MIH) Adrenocortical Mitotane (o,p′-DDD) SuppressantAminoglutethimide Hormones and Progestins Hydroxyprogesterone caproateAntagonists Medroxyprogesterone acetate Megestrol acetate EstrogensDiethylstilbestrol Ethinyl estradiol Antiestrogen Tamoxifen AndrogensTestosterone propionate Fluoxymesterone Antiandrogen FlutamideGonadotropin- Leuprolide releasing Hormone analog

The agents tested in the present methods can be a single agent or acombination of agents. For example, the present methods can be used todetermine whether a single chemotherapeutic agent, such as methotrexate,can be used to treat a cancer or whether a combination of two or moreagents can be used in combination with an NAE inhibitor (e.g., MLN4924).Useful combinations can include agents that have different mechanisms ofaction, e.g., the use of an anti-mitotic agent in combination with analkylating agent and an NAE inhibitor.

The agents disclosed herein may be administered by any route, includingintradermally, subcutaneously, orally, intraarterially or intravenously.In one embodiment, administration will be by the intravenous route.Parenteral administration can be provided in a bolus or by infusion.

The concentration of a disclosed compound in a pharmaceuticallyacceptable mixture will vary depending on several factors, including thedosage of the compound to be administered, the pharmacokineticcharacteristics of the compound(s) employed, and the route ofadministration. The agent may be administered in a single dose or inrepeat doses. Treatments may be administered daily or more frequentlydepending upon a number of factors, including the overall health of apatient, and the formulation and route of administration of the selectedcompound(s).

Screens for NAE Inhibitors

The invention provides methods (also referred to herein as “screeningassays”) for identifying modulators, i.e., candidate or test compoundsor agents (e.g., proteins, peptides, peptidomimetics, peptoids, smallmolecules or other drugs) which bind to NAE, or other E1 enzyme variantproteins, have a stimulatory or inhibitory effect on, for example, NAE,or other E1 enzyme expression or enzyme activity, or have a stimulatoryor inhibitory effect on, for example, the expression or activity of aNAE, or other E1 enzyme substrate or proteins in the E1 enzyme pathway,e.g., in the NAE pathway, e.g. with a relationship to the activity of acullin ring ligase. Compounds thus identified can be used to modulatethe activity of target gene products (e.g., NAE, or other E1 enzymegenes) in a therapeutic protocol, to elaborate the biological functionof the target gene product, or to identify compounds that disrupt NAE,or other E1 enzyme pathway interactions.

In one embodiment the invention provides a method of identifying acompound as an NAE inhibitor, e.g., as an agent that modulates the drugresistance of a cell, by first contacting a cell comprising at least onemutation in at least one marker gene with a test compound and thenmeasuring the viability of the cell or the inhibition of the growth ofthe cell. In some embodiments, the cell comprises a resistance geneidentified in Table 3. In other embodiments, the cell comprises asensitivity gene identified in Table 3. The effect of the NAE inhibitorcan be compared to a control cell not exposed to the compound. In someembodiments, the effect of an agent on a cell comprising a sensitivitymarker gene can be compared with the effect of an agent on a cellcomprising a resistance marker gene (see, e.g., Table 3). The compoundis identified as modulator of drug resistance or an NAE inhibitor agentwhen the cell viability or cell growth is decreased. The compoundsidentified as an NAE inhibitor, e.g., as modulating resistance, that areidentified in the foregoing methods are also included within theinvention.

Detection Methods

A general principle of prognostic assays involves preparing a sample orreaction mixture that may contain a marker, and a probe, underappropriate conditions and for a time sufficient to allow the marker andprobe to interact and bind, thus forming a complex that can be removedand/or detected in the reaction mixture. These assays can be conductedin a variety of ways.

For example, one method to conduct such an assay would involve anchoringthe marker or probe onto a solid phase support, also referred to as asubstrate, and detecting target marker/probe complexes anchored on thesolid phase at the end of the reaction. In one embodiment of such amethod, a sample from a subject, which is to be assayed for presenceand/or concentration of marker, can be anchored onto a carrier or solidphase support. In another embodiment, the reverse situation is possible,in which the probe can be anchored to a solid phase and a sample from asubject can be allowed to react as an unanchored component of the assay.One example of such an embodiment includes use of an array or chip whichcontains a predictive marker or marker set anchored for expressionanalysis of the sample.

There are many established methods for anchoring assay components to asolid phase. These include, without limitation, marker or probemolecules which are immobilized through conjugation of biotin andstreptavidin. Such biotinylated assay components can be prepared frombiotin-NHS (N-hydroxy-succinimide) using techniques known in the art(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). In certain embodiments, the surfaces with immobilized assaycomponents can be prepared in advance and stored.

Other suitable carriers or solid phase supports for such assays includeany material capable of binding the class of molecule to which themarker or probe belongs. Well-known supports or carriers include, butare not limited to, glass, polystyrene, nylon, polypropylene, nylon,polyethylene, dextran, amylases, natural and modified celluloses,polyacrylamides, gabbros, and magnetite. One skilled in the art willknow many other suitable carriers for binding antibody or antigen, andwill be able to adapt such support for use with the present invention.For example, protein isolated from cells can be run on a polyacrylamidegel electrophoresis and immobilized onto a solid phase support such asnitrocellulose. The support can then be washed with suitable buffersfollowed by treatment with the detectably labeled antibody. The solidphase support can then be washed with the buffer a second time to removeunbound antibody. The amount of bound label on the solid support canthen be detected by conventional means.

In order to conduct assays with the above mentioned approaches, thenon-immobilized component is added to the solid phase upon which thesecond component is anchored. After the reaction is complete,uncomplexed components may be removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized uponthe solid phase. The detection of marker/probe complexes anchored to thesolid phase can be accomplished in a number of methods outlined herein.

In an embodiment, the probe, when it is the unanchored assay component,can be labeled for the purpose of detection and readout of the assay,either directly or indirectly, with detectable labels discussed hereinand which are well-known to one skilled in the art. The term “labeled”,with regard to the probe (e.g., nucleic acid or antibody), is intendedto encompass direct labeling of the probe by coupling (i.e., physicallylinking) a detectable substance to the probe, as well as indirectlabeling of the probe by reactivity with another reagent that isdirectly labeled. An example of indirect labeling includes detection ofa primary antibody using a fluorescently labeled secondary antibody. Itis also possible to directly detect marker/probe complex formationwithout further manipulation or labeling of either component (marker orprobe), for example by utilizing the technique of fluorescence energytransfer (FET, see, for example, Lakowicz et al., U.S. Pat. No.5,631,169; Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). Afluorophore label on the first, ‘donor’ molecule is selected such that,upon excitation with incident light of appropriate wavelength, itsemitted fluorescent energy will be absorbed by a fluorescent label on asecond ‘acceptor’ molecule, which in turn is able to fluoresce due tothe absorbed energy. Alternately, the ‘donor’ protein molecule maysimply utilize the natural fluorescent energy of tryptophan residues.Labels are chosen that emit different wavelengths of light, such thatthe ‘acceptor’ molecule label may be differentiated from that of the‘donor’. Since the efficiency of energy transfer between the labels isrelated to the distance separating the molecules, spatial relationshipsbetween the molecules can be assessed. In a situation in which bindingoccurs between the molecules, the fluorescent emission of the ‘acceptor’molecule label in the assay should be maximal. An FET binding event canbe conveniently measured through standard fluorometric detection meanswell known in the art (e.g., using a fluorimeter).

In another embodiment, determination of the ability of a probe torecognize a marker can be accomplished without labeling either assaycomponent (probe or marker) by utilizing a technology such as real-timeBiomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. andUrbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995)Curr. Opin. Struct. Biol. 5:699-705). As used herein, “BIA” or “surfaceplasmon resonance” is a technology for studying biospecific interactionsin real time, without labeling any of the interactants (e.g., BIACORE™).Changes in the mass at the binding surface (indicative of a bindingevent) result in alterations of the refractive index of light near thesurface (the optical phenomenon of surface plasmon resonance (SPR)),resulting in a detectable signal which can be used as an indication ofreal-time reactions between biological molecules.

Alternatively, in another embodiment, analogous diagnostic andprognostic assays can be conducted with marker and probe as solutes in aliquid phase. In such an assay, the complexed marker and probe areseparated from uncomplexed components by any of a number of standardtechniques, including but not limited to: differential centrifugation,chromatography, electrophoresis and immunoprecipitation. In differentialcentrifugation, marker/probe complexes may be separated from uncomplexedassay components through a series of centrifugal steps, due to thedifferent sedimentation equilibria of complexes based on their differentsizes and densities (see, for example, Rivas, G., and Minton, A. P.(1993) Trends Biochem Sci. 18:284-7). Standard chromatographictechniques also can be utilized to separate complexed molecules fromuncomplexed ones. For example, gel filtration chromatography separatesmolecules based on size, and through the utilization of an appropriategel filtration resin in a column format, for example, the relativelylarger complex may be separated from the relatively smaller uncomplexedcomponents. Similarly, the relatively different charge properties of themarker/probe complex as compared to the uncomplexed components may beexploited to differentiate the complex from uncomplexed components, forexample through the utilization of ion-exchange chromatography resins.Such resins and chromatographic techniques are well known to one skilledin the art (see, e.g., Heegaard, N. H. (1998) J. Mol. Recognit.11:141-8; Hage, D. S., and Tweed, S. A. (1997) J. Chromatogr. B. Biomed.Sci. Appl. 699:499-525). Gel electrophoresis may also be employed toseparate complexed assay components from unbound components (see, e.g.,Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley& Sons, New York, 1987-1999). In this technique, protein or nucleic acidcomplexes are separated based on size or charge, for example. In someembodiments, non-denaturing gel matrix materials and conditions in theabsence of reducing agent are used in order to maintain the bindinginteraction during the electrophoretic process. Appropriate conditionsto the particular assay and components thereof will be well known to oneskilled in the art.

The isolated mRNA can be used in hybridization or amplification assaysthat include, but are not limited to, Southern or Northern analyses,polymerase chain reaction and TAQMAN® gene expression assays (AppliedBiosystems, Foster City, Calif.) and probe arrays. One diagnostic methodfor the detection of mRNA levels involves contacting the isolated mRNAwith a nucleic acid molecule (probe, e.g., a hybridization probe) thatcan hybridize to the mRNA encoded by the gene or mutant being detected.In some embodiments, nucleic acids comprising mutations of marker genescan be used as probes or primers. The nucleic acid probes or primers ofthe invention can be single stranded DNA (e.g., an oligonucleotide),double stranded DNA (e.g., double stranded oligonucleotide) or RNA.Primers of the invention refer to nucleic acids which hybridize to anucleic acid sequence which is adjacent to the region of interest andcan be extended over a region of interest, e.g., in a primer extensionor amplification reaction, or which covers the region of interest, e.g.,a nucleic acid region comprising a marker gene or mutation thereof. Anucleic acid probe can be, for example, a full-length cDNA, or a portionthereof, such as an oligonucleotide of at least 7, 15, 20, 25, 30, 50,75, 100, 125, 150, 175, 200, 250 or 500 or more consecutive nucleotidesof the marker nucleic acid sequence or complement thereof and sufficientto specifically hybridize under stringent conditions to a mRNA orgenomic DNA encoding a marker of the present invention or a complementthereof. The exact length of the nucleic acid probe will depend on manyfactors that are routinely considered and practiced by the skilledartisan. Nucleic acid probes of the invention may be prepared bychemical synthesis using any suitable methodology known in the art, maybe produced by recombinant technology, or may be derived from abiological sample, for example, by restriction digestion. Other suitableprobes for use in the diagnostic assays of the invention are describedherein. The probe can comprise a label group attached thereto, e.g., aradioisotope, a fluorescent compound, an enzyme, an enzyme co-factor, ahapten, a sequence tag, a protein or an antibody. The nucleic acids canbe modified at the base moiety, at the sugar moiety, or at the phosphatebackbone. An example of a nucleic acid label is incorporated usingSUPER™ Modified Base Technology (Nanogen, Bothell, Wash., see U.S. Pat.No. 7,045,610). The level of expression can be measured as generalnucleic acid levels, e.g., after measuring the amplified DNA levels(e.g. using a DNA intercalating dye, e.g., the SYBR green dye (QiagenInc., Valencia, Calif.) or as specific nucleic acids, e.g., using aprobe based design, with the probes labeled. TAQMAN® assay formats canuse the probe-based design to increase specificity and signal-to-noiseratio.

Such primers or probes can be used as part of a diagnostic test kit foridentifying cells or tissues which express the protein, such as bymeasuring amounts of a nucleic acid molecule transcribed in a sample ofcells from a subject, e.g., detecting transcript, mRNA levels ordetermining whether a gene encoding the protein has been mutated ordeleted. Hybridization of an RNA or a cDNA with the nucleic acid probecan indicate that the marker in question is being expressed. Theinvention further encompasses detecting nucleic acid molecules thatdiffer, due to degeneracy of the genetic code, from the nucleotidesequence of nucleic acids encoding a marker protein (e.g., proteinhaving the sequence of the SEQ ID NOs:3, 6, 10, 11, 14, 17, 20, 23 or26), and thus encode the same protein. It will be appreciated by thoseskilled in the art that DNA sequence polymorphisms that lead to changesin the amino acid sequence can exist within a population (e.g., thehuman population). Such genetic polymorphisms can exist amongindividuals within a population due to natural allelic variation. Anallele is one of a group of genes which occur alternatively at a givengenetic locus. Such natural allelic variations can typically result in1-5% variance in the nucleotide sequence of a given gene. Alternativealleles can be identified by sequencing the gene of interest in a numberof different individuals, e.g., normal samples from individuals. Thiscan be readily carried out by using hybridization probes to identify thesame genetic locus in a variety of individuals. Detecting any and allsuch nucleotide variations and resulting amino acid polymorphisms orvariations that are the result of natural allelic variation and that donot alter the functional activity are intended to be within the scope ofthe invention. In addition, it will be appreciated that DNApolymorphisms that affect RNA expression levels can also exist that mayaffect the overall expression level of that gene (e.g., by affectingregulation or degradation).

As used herein, the term “hybridizes” is intended to describe conditionsfor hybridization and washing under which nucleotide sequences that aresignificantly identical or homologous to each other remain hybridized toeach other. In some embodiments, the conditions are such that sequencesat least about 70%, at least about 80%, at least about 85%, 90% or 95%identical to each other remain hybridized to each other for subsequentamplification and/or detection. Stringent conditions vary according tothe length of the involved nucleotide sequence but are known to thoseskilled in the art and can be found or determined based on teachings inCurrent Protocols in Molecular Biology, Ausubel et al., eds., John Wiley& Sons, Inc. (1995), sections 2, 4 and 6. Additional stringentconditions and formulas for determining such conditions can be found inMolecular Cloning: A Laboratory Manual, Sambrook et al., Cold SpringHarbor Press, Cold Spring Harbor, N.Y. (1989), chapters 7, 9 and 11. Anon-limiting example of stringent hybridization conditions for hybridsthat are at least 10 basepairs in length includes hybridization in 4×sodium chloride/sodium citrate (SSC), at about 65-70° C. (orhybridization in 4×SSC plus 50% formamide at about 42-50° C.) followedby one or more washes in 1×SSC, at about 65-70° C. A non-limitingexample of highly stringent hybridization conditions for such hybridsincludes hybridization in 1×SSC, at about 65-70° C. (or hybridization in1×SSC plus 50% formamide at about 42-50° C.) followed by one or morewashes in 0.3×SSC, at about 65-70° C. A non-limiting example of reducedstringency hybridization conditions for such hybrids includeshybridization in 4×SSC, at about 50-60° C. (or alternativelyhybridization in 6×SSC plus 50% formamide at about 40-45° C.) followedby one or more washes in 2×SSC, at about 50-60° C. Ranges intermediateto the above-recited values, e.g., at 65-70° C. or at 42-50° C. are alsointended to be encompassed by the present invention. Another example ofstringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65° C. A further example of stringenthybridization buffer is hybridization in 1 M NaCl, 50 mM2-(N-morpholino)ethanesulfonic acid (IVIES) buffer (pH 6.5), 0.5% sodiumsarcosine and 30% formamide. SSPE (1×SSPE is 0.15M NaCl, 10 mM NaH₂PO₄,and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1×SSC is 0.15MNaCl and 15 mM sodium citrate) in the hybridization and wash buffers;washes are performed for 15 minutes each after hybridization is completeThe hybridization temperature for hybrids anticipated to be less than 50base pairs in length should be 5-10° C. less than the meltingtemperature (T_(m)) of the hybrid, where T_(m) is determined accordingto the following equations. For hybrids less than 18 base pairs inlength, T_(m)(° C.)=2(# of A+T bases)+4(# of G+C bases). For hybridsbetween 18 and 49 base pairs in length, T_(m)(°C.)=81.5+16.6(log₁₀[Na⁺])+0.41(% G+C)−(600/N), where N is the number ofbases in the hybrid, and [Na⁺] is the concentration of sodium ions inthe hybridization buffer ([Na⁺] for 1×SSC=0.165 M). It will also berecognized by the skilled practitioner that additional reagents may beadded to hybridization and/or wash buffers to decrease non-specifichybridization of nucleic acid molecules to membranes, for example,nitrocellulose or nylon membranes, including but not limited to blockingagents (e.g., BSA or salmon or herring sperm carrier DNA), detergents(e.g., SDS), chelating agents (e.g., EDTA), Ficoll, polyvinylpyrrolidone(PVP) and the like. When using nylon membranes, in particular, anadditional non-limiting example of stringent hybridization conditions ishybridization in 0.25-0.5M NaH₂PO₄, 7% SDS at about 65° C., followed byone or more washes at 0.02M NaH₂PO₄, 1% SDS at 65° C., see e.g., Churchand Gilbert (1984) Proc. Natl. Acad. Sci. USA 81:1991-1995, (oralternatively 0.2×SSC, 1% SDS). A primer or nucleic acid probe can beused alone in a detection method, or a primer can be used together withat least one other primer or nucleic acid probe in a detection method.Primers can also be used to amplify at least a portion of a nucleicacid. Nucleic acid probes of the invention refer to nucleic acids whichhybridize to the region of interest and which are not further extended.For example, a nucleic acid probe is a nucleic acid which specificallyhybridizes to a mutant region of a biomarker, and which by hybridizationor absence of hybridization to the DNA of a patient or the type ofhybrid formed can be indicative of the presence or identity of themutation of the biomarker or the amount of marker activity.

In one format, the RNA is immobilized on a solid surface and contactedwith a probe, for example by running the isolated RNA on an agarose geland transferring the RNA from the gel to a membrane, such asnitrocellulose. In an alternative format, the nucleic acid probe(s) areimmobilized on a solid surface and the RNA is contacted with theprobe(s), for example, in an AFFYMETRIX® gene chip array or a SNP chip(Santa Clara, Calif.) or customized array using a marker set comprisingat least one marker indicative of treatment outcome. A skilled artisancan readily adapt known RNA and DNA detection methods for use indetecting the amount of the markers of the present invention. Forexample, the high density microarray or branched DNA assay can benefitfrom a higher concentration of tumor cell in the sample, such as asample which had been modified to isolate tumor cells as described inearlier sections. In a related embodiment, a mixture of transcribedpolynucleotides obtained from the sample is contacted with a substratehaving fixed thereto a polynucleotide complementary to or homologouswith at least a portion (e.g., at least 7, 10, 15, 20, 25, 30, 40, 50,100, 500, or more nucleotide residues) of a marker nucleic acid. Ifpolynucleotides complementary to or homologous with the marker aredifferentially detectable on the substrate (e.g., detectable usingdifferent chromophores or fluorophores, or fixed to different selectedpositions), then the levels of expression of a plurality of markers canbe assessed simultaneously using a single substrate (e.g., a “gene chip”microarray of polynucleotides fixed at selected positions). In anembodiment when a method of assessing marker expression is used whichinvolves hybridization of one nucleic acid with another, thehybridization can be performed under stringent hybridization conditions.

An alternative method for determining the amount of RNA corresponding toa marker of the present invention in a sample involves the process ofnucleic acid amplification, e.g., by RT-PCR (the experimental embodimentset forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chainreaction (Barmy, 1991, Proc. Natl. Acad. Sci. USA, 88:189-193), selfsustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad.Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh etaL, 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi et al., 1988, Bio/Technology 6:1197), rolling circlereplication (Lizardi et al., U.S. Pat. No. 5,854,033) or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques well known to those of skill in theart. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers. As used herein, amplification primers are defined as being apair of nucleic acid molecules that can anneal to 5′ or 3′ regions of agene (plus and minus strands, respectively, or vice-versa) and contain ashort region in between. In general, amplification primers are fromabout 10 to about 30 nucleotides in length and flank a region from about50 to about 200 nucleotides in length. Under appropriate conditions andwith appropriate reagents, such primers permit the amplification of anucleic acid molecule comprising the nucleotide sequence flanked by theprimers.

For in situ methods, RNA does not need to be isolated from the cellsprior to detection. In such methods, a cell or tissue sample isprepared/processed using known histological methods. The sample is thenimmobilized on a support, typically a glass slide, and then contactedwith a probe that can hybridize to RNA that encodes the marker.

In another embodiment of the present invention, a polypeptidecorresponding to a marker is detected. In some embodiments, an agent fordetecting a polypeptide of the invention is an antibody capable ofbinding to a polypeptide corresponding to a marker of the invention. Inrelated embodiments, the antibody has a detectable label. Antibodies canbe polyclonal, or monoclonal. An intact antibody, or a fragment thereof(e.g., Fab or F(ab′)₂) can be used.

A variety of formats can be employed to determine whether a samplecontains a protein that binds to a given antibody. Examples of suchformats include, but are not limited to, enzyme immunoassay (EIA),radioimmunoassay (RIA), Western blot analysis and enzyme linkedimmunoabsorbent assay (ELISA). A skilled artisan can readily adapt knownprotein/antibody detection methods for use in determining whether Bcells express a marker of the present invention.

Another method for determining the level of a polypeptide correspondingto a marker is mass spectrometry. For example, intact proteins orpeptides, e.g., tryptic peptides can be analyzed from a sample, e.g., ablood sample, a lymph sample or other sample, containing one or morepolypeptide markers. The method can further include treating the sampleto lower the amounts of abundant proteins, e.g., serum albumin, toincrease the sensitivity of the method. For example, liquidchromatography can be used to fractionate the sample so portions of thesample can be analyzed separately by mass spectrometry. The steps can beperformed in separate systems or in a combined liquidchromatography/mass spectrometry system (LC/MS, see for example, Liao,et al. (2004) Arthritis Rheum. 50:3792-3803). The mass spectrometrysystem also can be in tandem (MS/MS) mode. The charge state distributionof the protein or peptide mixture can be acquired over one or multiplescans and analyzed by statistical methods, e.g. using the retention timeand mass-to-charge ratio (m/z) in the LC/MS system, to identify proteinsexpressed at statistically significant levels differentially in samplesfrom patients responsive or non-responsive to NAE inhibition therapy.Examples of mass spectrometers which can be used are an ion trap system(ThermoFinnigan, San Jose, Calif.) or a quadrupole time-of-flight massspectrometer (Applied Biosystems, Foster City, Calif.). The method canfurther include the step of peptide mass fingerprinting, e.g. in amatrix-assisted laser desorption ionization with time-of-flight(MALDI-TOF) mass spectrometry method. The method can further include thestep of sequencing one or more of the tryptic peptides. Results of thismethod can be used to identify proteins from primary sequence databases,e.g., maintained by the National Center for Biotechnology Information,Bethesda, Md., or the Swiss Institute for Bioinformatics, Geneva,Switzerland, and based on mass spectrometry tryptic peptide m/z basepeaks.

Electronic Apparatus Readable Arrays

Electronic apparatus, including readable arrays comprising at least onepredictive marker of the present invention is also contemplated for usein conjunction with the methods of the invention. As used herein,“electronic apparatus readable media” refers to any suitable medium forstoring, holding or containing data or information that can be read andaccessed directly by an electronic apparatus. As used herein, the term“electronic apparatus” is intended to include any suitable computing orprocessing apparatus or other device configured or adapted for storingdata or information. Examples of electronic apparatus suitable for usewith the present invention and monitoring of the recorded informationinclude stand-alone computing apparatus; networks, including a localarea network (LAN), a wide area network (WAN) Internet, Intranet, andExtranet; electronic appliances such as personal digital assistants(PDAs), cellular phone, pager and the like; and local and distributedprocessing systems. As used herein, “recorded” refers to a process forstoring or encoding information on the electronic apparatus readablemedium. Those skilled in the art can readily adopt any of the presentlyknown methods for recording information on known media to generatemanufactures comprising the markers of the present invention.

For example, microarray systems are well known and used in the art forassessment of samples, whether by assessment gene expression (e.g., DNAdetection, RNA detection, protein detection), or metabolite production,for example. Microarrays for use according to the invention include oneor more probes of predictive marker(s) of the invention characteristicof response and/or non-response to a therapeutic regimen as describedherein. In one embodiment, the microarray comprises one or more probescorresponding to one or more of markers selected from the groupconsisting of markers which demonstrate increased expression in shortterm survivors, and genes which demonstrate increased expression in longterm survivors in patients. A number of different microarrayconfigurations and methods for their production are known to those ofskill in the art and are disclosed, for example, in U.S. Pat. Nos.5,242,974; 5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,429,807;5,436,327; 5,445,934; 5,556,752; 5,405,783; 5,412,087; 5,424,186;5,429,807; 5,436,327; 5,472,672; 5,527,681; 5,529,756; 5,545,531;5,554,501; 5,561,071; 5,571,639; 5,593,839; 5,624,711; 5,700,637;5,744,305; 5,770,456; 5,770,722; 5,837,832; 5,856,101; 5,874,219;5,885,837; 5,919,523; 5,981,185; 6,022,963; 6,077,674; 6,156,501;6,261,776; 6,346,413; 6,440,677; 6,451,536; 6,576,424; 6,610,482;5,143,854; 5,288,644; 5,324,633; 5,432,049; 5,470,710; 5,492,806;5,503,980; 5,510,270; 5,525,464; 5,547,839; 5,580,732; 5,661,028;5,848,659; and 5,874,219; Shena, et al. (1998), Tibtech 16:301; Dugganet al. (1999) Nat. Genet. 21:10; Bowtell et al. (1999) Nat. Genet.21:25; Lipshutz et al. (1999) Nature Genet. 21:20-24, 1999; Blanchard,et al. (1996) Biosensors and Bioelectronics, 11:687-90; Maskos, et al.,(1993) Nucleic Acids Res. 21:4663-69; Hughes, et al. (2001) Nat.Biotechnol. 19:342, 2001; each of which are herein incorporated byreference. A tissue microarray can be used for protein identification(see Hans et al. (2004) Blood 103:275-282). A phage-epitope microarraycan be used to identify one or more proteins in a sample based onwhether the protein or proteins induce auto-antibodies in the patient(Bradford et al. (2006) Urol. Oncol. 24:237-242).

A microarray thus comprises one or more probes corresponding to one ormore markers identified herein, e.g., those indicative of treatmentoutcome, e.g., to identify wild type marker genes, normal allelicvariants and mutations of marker genes. The microarray can compriseprobes corresponding to, for example, at least 2, at least 3, at least4, at least 5, at least 10, at least 15, at least 20, at least 25, atleast 30, at least 35, at least 40, at least 45, at least 50, at least75, or at least 100, biomarkers and/or mutations thereof indicative oftreatment outcome. The microarray can comprise probes corresponding toone or more biomarkers as set forth herein. Still further, themicroarray may comprise complete marker sets as set forth herein andwhich may be selected and compiled according to the methods set forthherein. The microarray can be used to assay expression of one or morepredictive markers or predictive marker sets in the array. In oneexample, the array can be used to assay more than one predictive markeror marker set expression in a sample to ascertain an expression profileof markers in the array. In this manner, up to about 44,000 markers canbe simultaneously assayed for expression. This allows an expressionprofile to be developed showing a battery of markers specificallyexpressed in one or more samples. Still further, this allows anexpression profile to be developed to assess treatment outcome.

The array is also useful for ascertaining differential expressionpatterns of one or more markers in normal and abnormal (e.g., sample,e.g., tumor) cells. This provides a battery of markers that could serveas a tool for ease of identification of treatment outcome of patients.Further, the array is useful for ascertaining expression of referencemarkers for reference expression levels. In another example, the arraycan be used to monitor the time course of expression of one or moremarkers in the array.

In addition to such qualitative determination, the invention allows thequantification of marker expression. Thus, predictive markers can begrouped on the basis of marker sets or outcome indications by the amountof the marker in the sample. This is useful, for example, inascertaining the outcome of the sample by virtue of scoring the amountsaccording to the methods provided herein.

The array is also useful for ascertaining the effect of the expressionof a marker on the expression of other predictive markers in the samecell or in different cells. This provides, for example, a selection ofalternate molecular targets for therapeutic intervention if patient ispredicted to have an unfavorable outcome.

Reagents and Kits

The invention also encompasses kits for detecting the presence of apolypeptide or nucleic acid corresponding to a marker of the inventionin a biological sample (e.g. a bone marrow sample, tumor biopsy or areference sample). Such kits can be used to determine mutational statusof at least one marker gene to assess treatment outcome, e.g., determineif a subject can have a favorable outcome, e.g., after NAE inhibitortreatment. For example, the kit can comprise a labeled compound or agentcapable of detecting a genomic DNA segment, a polypeptide or atranscribed RNA corresponding to a marker of the invention or a mutationof a marker gene in a biological sample and means for determining theamount of the genomic DNA segment, the polypeptide or RNA in the sample.Suitable reagents for binding with a marker protein include antibodies,antibody derivatives, antibody fragments, and the like. Suitablereagents for binding with a marker nucleic acid (e.g., a genomic DNA, anmRNA, a spliced mRNA, a cDNA, or the like) include complementary nucleicacids. The label can be directly attached to the marker binding agent,e.g., probe, e.g., nucleic acid reagent such as a probe or primer orprotein reagent, such as a specific binding agent or antibody, or asecondary reagent can comprise a label for indirect labeling. The kitcan also contain a control or reference sample or a series of control orreference samples which can be assayed and compared to the test sample.For example, the kit may have a positive control sample, e.g., includingone or more markers or mutations described herein, or reference markers,e.g. housekeeping markers to standardize the assay among samples ortimepoints or reference genomes, e.g., form subjects without tumor e.g.,to establish diploid copy number baseline or reference expression levelof a marker. By way of example, the kit may comprise fluids (e.g.,buffer) suitable for annealing complementary nucleic acids or forbinding an antibody with a protein with which it specifically binds andone or more sample compartments. The kit of the invention may optionallycomprise additional components useful for performing the methods of theinvention, e.g., a sample collection vessel, e.g., a tube, andoptionally, means for optimizing the amount of marker detected, forexample if there may be time or adverse storage and handling conditionsbetween the time of sampling and the time of analysis. For example, thekit can contain means for increasing the number of tumor cells in thesample, as described above, a buffering agent, a preservative, astabilizing agent or additional reagents for preparation of cellularmaterial or probes for use in the methods provided; and detectablelabel, alone or conjugated to or incorporated within the providedprobe(s). In one exemplary embodiment, a kit comprising a samplecollection vessel can comprise e.g., a tube comprising anti-coagulantand/or stabilizer, e.g., an RNA stabilizer, as described above, or knownto those skilled in the art. The kit can further comprise componentsnecessary for detecting the detectable label (e.g., an enzyme or asubstrate). For marker sets, the kit can comprise a marker set array orchip for use in detecting the biomarkers. Kits also can includeinstructions for interpreting the results obtained using the kit. Thekit can contain reagents for detecting one or more biomarkers, e.g., 2,3, 4, 5, or more biomarkers described herein.

In one embodiment, the kit comprises a probe to detect at least onebiomarker, e.g., a marker indicative of treatment outcome (e.g., uponNAE inhibitor treatment). In an exemplary embodiment, the kit comprisesa nucleic acid probe to detect a marker gene selected from the groupconsisting of SEQ ID NO: 1, 2, 4, 5, 7, 8, 9, 12, 13, 15, 16, 18, 19,21, 22, 24, 25 or a sequence on chromosome 22q from base pair 29999545to 30094589, chromosome 18q from base pair 48556583 to 48611412,chromosome Xp from base pair 44732423 to 44971847, chromosome 4q frombase pair 153242410 to Ser. No. 15/345,6172, chromosome 17p from basepair 7571720 to 7590868, chromosome 9p from base pair 21967751 to21994490, or a complement of any of the foregoing or SEQ ID NO: 3, 6,10, 11, 14, 17, 20, 23 and/or 26. In some embodiments, the kit comprisesa probe to detect a marker selected from the group consisting of NF2,SMAD4, KDM6A, FBXW7, TP53, CDKN2A, CDKN2A_p14 and APC. In otherembodiments, the kit comprises a probe to detect a mutation in a markergene selected from the group consisting of NF2, SMAD4, KDM6A, FBXW7,TP53, CDKN2A, CDKN2A_p14 and APC. In an embodiment, a kit comprisesprobes to detect a marker set comprising two or more markers from thegroup consisting of NF2, SMAD4, KDM6A, FBXW7, TP53, CDKN2A, CDKN2A_p14and APC. In another embodiment, a kit comprises a probe to detect FBXW7in cancer of the uterus or cervix. In an embodiment, a kit comprises aprobe to detect TP53 in cancer of the intestine, breast, lung, head andneck, cervix or skin. In an embodiment, a kit comprises a probe todetect TPC and APC in cancer of the intestine. In an embodiment, a kitcomprises a probe to detect CDKN2A_p14 in cancer of the skin or centralnervous system. In an embodiment, a kit comprises a probe to detectCDKN2A in cancer of the head and neck or skin. In an embodiment, a kitcomprises a probe to detect SMAD4 in cancer of the head and neck. Inrelated embodiments, the kit comprises a nucleic acid probe comprisingor derived from (e.g., a fragment, mutant or variant (e.g., homologousor complementary) thereof) a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 1, 2, 4, 5, 7, 8, 9, 12, 13, 15, 16, 18,19, 21, 22, 24, and 25. For kits comprising nucleic acid probes, e.g.,oligonucleotide-based kits, the kit can comprise, for example: one ormore nucleic acid reagents such as an oligonucleotide (labeled ornon-labeled) which hybridizes to a nucleic acid sequence correspondingto a marker of the invention, optionally fixed to a substrate; and canoptionally further comprise labeled oligonucleotides not bound with asubstrate, a primer, a pair of PCR primers, e.g., useful for amplifyinga nucleic acid molecule corresponding to a marker of the invention,molecular beacon probes, a marker set comprising oligonucleotides whichhybridize to at least two nucleic acid sequences corresponding tomarkers of the invention, and the like. The kit can contain anRNA-stabilizing agent.

For kits comprising protein probes, e.g., ligand or antibody-based kits,the kit can comprise, for example: (1) a first antibody (e.g., attachedto a solid support) which binds to a polypeptide corresponding to amarker of the invention; and, optionally, (2) a second, differentantibody which binds to either the polypeptide or the first antibody andis conjugated to a detectable label. The kit can contain a proteinstabilizing agent. The kit can contain reagents to reduce the amount ofnon-specific binding of non-biomarker material from the sample to theprobe. Examples of reagents to reduce non-specific binding includenonioinic detergents, non-specific protein containing solutions, such asthose containing albumin or casein, or other substances known to thoseskilled in the art.

An isolated polypeptide corresponding to a predictive marker of theinvention, or a fragment or mutant thereof, can be used as an immunogento generate antibodies using standard techniques for polyclonal andmonoclonal antibody preparation. For example, an immunogen typically isused to prepare antibodies by immunizing a suitable (i.e.,immunocompetent) subject such as a rabbit, goat, mouse, or other mammalor vertebrate. In still a further aspect, the invention providesmonoclonal antibodies or antigen binding fragments thereof, whichantibodies or fragments specifically bind to a polypeptide comprising anamino acid sequence selected from the group consisting of the amino acidsequences of the present invention, an amino acid sequence encoded bythe cDNA of the present invention, a fragment of at least 8, 10, 12, 15,20 or 25 consecutive amino acid residues of an amino acid sequence ofthe present invention, an amino acid sequence which is at least 95%,96%, 97%, 98% or 99% identical to an amino acid sequence of the presentinvention (wherein the percent identity is determined using the ALIGNprogram of the GCG software package with a PAM120 weight residue table,a gap length penalty of 12, and a gap penalty of 4) and an amino acidsequence which is encoded by a nucleic acid molecule which hybridizes toa nucleic acid molecule consisting of the nucleic acid molecules of thepresent invention, or a complement thereof, under conditions ofhybridization of 6×SSC at 45° C. and washing in 0.2×SSC, 0.1% SDS at 65°C. The monoclonal antibodies can be human, humanized, chimeric and/ornon-human antibodies. An appropriate immunogenic preparation cancontain, for example, recombinantly-expressed or chemically-synthesizedpolypeptide. The preparation can further include an adjuvant, such asFreund's complete or incomplete adjuvant, or a similar immunostimulatoryagent.

Methods for making human antibodies are known in the art. One method formaking human antibodies employs the use of transgenic animals, such as atransgenic mouse. These transgenic animals contain a substantial portionof the human antibody producing genome inserted into their own genomeand the animal's own endogenous antibody production is rendereddeficient in the production of antibodies. Methods for making suchtransgenic animals are known in the art. Such transgenic animals can bemade using XENOMOUSE™ technology or by using a “minilocus” approach.Methods for making XENOMICE™ are described in U.S. Pat. Nos. 6,162,963,6,150,584, 6,114,598 and 6,075,181, which are incorporated herein byreference. Methods for making transgenic animals using the “minilocus”approach are described in U.S. Pat. Nos. 5,545,807, 5,545,806 and5,625,825; also see International Publication No. WO93/12227, which areeach incorporated herein by reference.

Antibodies include immunoglobulin molecules and immunologically activeportions of immunoglobulin molecules, i.e., molecules that contain anantigen binding site which specifically binds an antigen, such as apolypeptide of the invention, e.g., an epitope of a polypeptide of theinvention. A molecule which specifically binds to a given polypeptide ofthe invention is a molecule which binds the polypeptide, but does notsubstantially bind other molecules in a sample, e.g., a biologicalsample, which naturally contains the polypeptide. For example,antigen-binding fragments, as well as full-length monomeric, dimeric ortrimeric polypeptides derived from the above-described antibodies arethemselves useful. Useful antibody homologs of this type include (i) aFab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the VH and CH1 domains; (iv) a Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody, (v)a dAb fragment (Ward et al., Nature 341:544-546 (1989)), which consistsof a VH domain; (vii) a single domain functional heavy chain antibody,which consists of a VHH domain (known as a nanobody) see e.g.,Cortez-Retamozo, et al., Cancer Res. 64: 2853-2857 (2004), andreferences cited therein; and (vii) an isolated complementaritydetermining region (CDR), e.g., one or more isolated CDRs together withsufficient framework to provide an antigen binding fragment.Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al.Science 242:423-426 (1988); and Huston et al. Proc. Natl. Acad. Sci. USA85:5879-5883 (1988). Such single chain antibodies are also intended tobe encompassed within the term “antigen-binding fragment” of anantibody. These antibody fragments are obtained using conventionaltechniques known to those with skill in the art, and the fragments arescreened for utility in the same manner as are intact antibodies.Antibody fragments, such as Fv, F(ab′)₂ and Fab may be prepared bycleavage of the intact protein, e.g. by protease or chemical cleavage.The invention provides polyclonal and monoclonal antibodies. Syntheticand genetically engineered variants (See U.S. Pat. No. 6,331,415) of anyof the foregoing are also contemplated by the present invention.Polyclonal and monoclonal antibodies can be produced by a variety oftechniques, including conventional murine monoclonal antibodymethodology e.g., the standard somatic cell hybridization technique ofKohler and Milstein, Nature 256: 495 (1975) the human B cell hybridomatechnique (see Kozbor et al., 1983, Immunol. Today 4:72), theEBV-hybridoma technique (see Cole et al., pp. 77-96 In MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., 1985) or triomatechniques. See generally, Harlow, E. and Lane, D. (1988) Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; and Current Protocols in Immunology, Coligan et al. ed.,John Wiley & Sons, New York, 1994. For diagnostic applications, theantibodies can be monoclonal antibodies, e.g., generated in mouse, rat,or rabbit. Additionally, for use in in vivo applications the antibodiesof the present invention can be human or humanized antibodies. Hybridomacells producing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindthe polypeptide of interest, e.g., using a standard ELISA assay.

If desired, the antibody molecules can be harvested or isolated from thesubject (e.g., from the blood or serum of the subject) and furtherpurified by well-known techniques, such as protein A chromatography toobtain the IgG fraction. Alternatively, antibodies specific for aprotein or polypeptide of the invention can be selected or (e.g.,partially purified) or purified by, e.g., affinity chromatography toobtain substantially purified and purified antibody. By a substantiallypurified antibody composition is meant, in this context, that theantibody sample contains at most only 30% (by dry weight) ofcontaminating antibodies directed against epitopes other than those ofthe desired protein or polypeptide of the invention, and at most 20%, atmost 10%, or at most 5% (by dry weight) of the sample is contaminatingantibodies. A purified antibody composition means that at least 99% ofthe antibodies in the composition are directed against the desiredprotein or polypeptide of the invention.

An antibody directed against a polypeptide corresponding to a marker ofthe invention (e.g., a monoclonal antibody) can be used to detect themarker (e.g., in a cellular sample) in order to evaluate the level andpattern of expression of the marker. The antibodies can also be useddiagnostically to monitor protein levels in tissues or body fluids (e.g.in a blood sample or urine) as part of a clinical testing procedure,e.g., to, for example, determine the efficacy of a given treatmentregimen. Detection can be facilitated by coupling the antibody to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

Accordingly, in one aspect, the invention provides substantiallypurified antibodies or fragments thereof, and non-human antibodies orfragments thereof, which antibodies or fragments specifically bind to apolypeptide comprising an amino acid sequence encoded by a markeridentified herein. The substantially purified antibodies of theinvention, or fragments thereof, can be human, non-human, chimericand/or humanized antibodies.

In another aspect, the invention provides non-human antibodies orfragments thereof, which antibodies or fragments specifically bind to apolypeptide comprising an amino acid sequence which is encoded by anucleic acid molecule of a predictive marker of the invention. Suchnon-human antibodies can be goat, mouse, sheep, horse, chicken, rabbit,or rat antibodies. Alternatively, the non-human antibodies of theinvention can be chimeric and/or humanized antibodies. In addition, thenon-human antibodies of the invention can be polyclonal antibodies ormonoclonal antibodies.

The substantially purified antibodies or fragments thereof mayspecifically bind to a signal peptide, a secreted sequence, anextracellular domain, a transmembrane or a cytoplasmic domain orcytoplasmic loop of a polypeptide of the invention. The substantiallypurified antibodies or fragments thereof, the non-human antibodies orfragments thereof, and/or the monoclonal antibodies or fragmentsthereof, of the invention specifically bind to a secreted sequence or anextracellular domain of the amino acid sequences of the presentinvention.

The invention also provides a kit containing an antibody of theinvention conjugated to a detectable substance, and instructions foruse. Still another aspect of the invention is a diagnostic compositioncomprising a probe of the invention and a pharmaceutically acceptablecarrier. In one embodiment, the diagnostic composition contains anantibody of the invention, a detectable moiety, and a pharmaceuticallyacceptable carrier.

Sensitivity Assays

A sample of cancerous cells is obtained from a patient. An expressionlevel is measured in the sample for a marker corresponding to at leastone of the markers described herein. A marker set can be utilizedcomprising markers identified described herein, and put together in amarker set using the methods described herein. Such analysis is used toobtain an expression profile of the tumor in the patient. Evaluation ofthe expression profile is then used to determine whether the patient isexpected to have a favorable outcome and would benefit from treatment,e.g., NAE inhibition therapy (e.g., treatment with a NAE inhibitor(e.g., MLN4924) alone, or in combination with additional agents)), or analternative agent expected to have a similar effect on survival.Evaluation of the expression profile can also be used to determinewhether a patient is expected to have an unfavorable outcome and wouldbenefit from a cancer therapy other than NAE inhibition therapy or wouldbenefit from an altered NAE inhibition therapy regimen. Evaluation caninclude use of one marker set prepared using any of the methods providedor other similar scoring methods known in the art (e.g., weightedvoting, combination of threshold features (CTF), Cox proportionalhazards analysis, principal components scoring, linear predictive score,K-nearest neighbor, etc), e.g., using expression values deposited withthe Gene Expression Omnibus (GEO) program at the National Center forBiotechnology Information (NCBI, Bethesda, Md.). Still further,evaluation can comprise use of more than one prepared marker set. A NAEinhibition therapy will be identified as appropriate to treat the cancerwhen the outcome of the evaluation demonstrates a favorable outcome or amore aggressive therapy regimen will be identified for a patient with anexpected unfavorable outcome.

In one aspect, the invention features a method of evaluating a patient,e.g., a patient with cancer, e.g. a hematological cancer (e.g., multiplemyeloma, leukemias, lymphoma, etc) or solid tumor cancer (e.g.,melanoma, esophageal cancer or bladder cancer) for treatment outcome.The method includes providing an evaluation of the expression of themarkers in a marker set of markers in the patient, wherein the markerset has the following properties: it includes a plurality of genes, eachof which is differentially expressed as between patients with identifiedoutcome and non-afflicted subjects and it contains a sufficient numberof differentially expressed markers such that differential amount (e.g.,as compared to a level in a non-afflicted reference sample) of each ofthe markers in the marker set in a subject is predictive of treatmentoutcome with no more than about 15%, about 10%, about 5%, about 2.5%, orabout 1% false positives (wherein false positive means predicting that apatient as responsive or non-responsive when the subject is not); andproviding a comparison of the amount of each of the markers in the setfrom the patient with a reference value, thereby evaluating the patient.

Examining the amount of one or more of the identified markers or markersets in a tumor sample taken from a patient during the course of NAEinhibition therapy, it is also possible to determine whether thetherapeutic agent is continuing to work or whether the cancer has becomenon-responsive (refractory) to the treatment protocol. For example, apatient receiving a treatment of MLN4924 would have tumor cells removedand monitored for the expression of a marker or marker set. If theprofile of the amount of one or more markers identified herein moretypifies favorable outcome in the presence of the agent, e.g., the NAEinhibitor, the treatment would continue. However, if the profile of theamount of one or more markers identified herein more typifiesunfavorable outcome in the presence of the agent, then the cancer mayhave become resistant to therapy, e.g., NAE inhibition therapy, andanother treatment protocol should be initiated to treat the patient. Forexample, the cancer may comprise a mutation in a marker gene associatedwith resistance to NAE inhibition.

Importantly, these determinations can be made on a patient-by-patientbasis or on an agent-by-agent (or combinations of agents). Thus, one candetermine whether or not a particular NAE inhibition therapy is likelyto benefit a particular patient or group/class of patients, or whether aparticular treatment should be continued.

Use of Information

In one method, information, e.g., about the mutational status of apatient's tumor, e.g., the patient's marker(s) characteristic, e.g.,size, sequence, composition or amount (e.g., the result of evaluating amarker or marker set described herein), or about whether a patient isexpected to have a favorable outcome, is provided (e.g., communicated,e.g., electronically communicated) to a third party, e.g., a hospital,clinic, a government entity, reimbursing party or insurance company(e.g., a life insurance company). For example, choice of medicalprocedure, whether to pay for a medical procedure, payment by areimbursing party, or cost for a service or insurance can be function ofthe information. E.g., the third party receives the information, makes adetermination based at least in part on the information, and optionallycommunicates the information or makes a choice of procedure, payment,level of payment, coverage, etc. based on the information. In themethod, informative expression level of a marker or a marker setselected from or derived from Table 1 and/or described herein isdetermined.

In one embodiment, a premium for insurance (e.g., life or medical) isevaluated as a function of information about one or more markerexpression levels, e.g., a marker or marker set, e.g., a level ofexpression associated with treatment outcome (e.g., the informativeamount). For example, premiums can be increased (e.g., by a certainpercentage) if the marker genes of a patient or a patient's marker setdescribed herein have different characteristic, e.g., size, sequence,composition or amount between an insured candidate (or a candidateseeking insurance coverage) and a reference value (e.g., a non-afflictedperson) or a reference sample, e.g., matched control. Premiums can alsobe scaled depending on the result of evaluating a marker or marker setdescribed herein. For example, premiums can be assessed to distributerisk, e.g., as a function of marker, e.g., the result of evaluating amarker or marker set described herein. In another example, premiums areassessed as a function of actuarial data that is obtained from patientsthat have known treatment outcomes.

Information about marker characteristic, e.g., size, sequence,composition or amount, e.g., the result of evaluating a marker or markerset described herein (e.g., the informative amount), can be used, e.g.,in an underwriting process for life insurance. The information can beincorporated into a profile about a subject. Other information in theprofile can include, for example, date of birth, gender, marital status,banking information, credit information, children, and so forth. Aninsurance policy can be recommended as a function of the information onmarker characteristic, e.g., size, sequence, composition or amount,e.g., the result of evaluating a marker or marker set described herein,along with one or more other items of information in the profile. Aninsurance premium or risk assessment can also be evaluated as functionof the marker or marker set information. In one implementation, pointsare assigned on the basis of expected treatment outcome.

In one embodiment, information about marker characteristic, e.g., size,sequence, composition or amount, e.g., the result of evaluating a markeror marker set described herein, is analyzed by a function thatdetermines whether to authorize the transfer of funds to pay for aservice or treatment provided to a subject (or make another decisionreferred to herein). For example, the results of analyzing acharacteristic, e.g., size, sequence, composition or amount of a markeror marker set described herein may indicate that a subject is expectedto have a favorable outcome, suggesting that a treatment course isneeded, thereby triggering an result that indicates or causesauthorization to pay for a service or treatment provided to a subject.In one example, informative characteristic, e.g., size, sequence,composition or amount of a marker or a marker set selected from orderived from Table 1 and/or described herein is determined and paymentis authorized if the informative amount identifies a favorable outcome.For example, an entity, e.g., a hospital, care giver, government entity,or an insurance company or other entity which pays for, or reimbursesmedical expenses, can use the result of a method described herein todetermine whether a party, e.g., a party other than the subject patient,will pay for services (e.g., a particular therapy) or treatment providedto the patient. For example, a first entity, e.g., an insurance company,can use the outcome of a method described herein to determine whether toprovide financial payment to, or on behalf of, a patient, e.g., whetherto reimburse a third party, e.g., a vendor of goods or services, ahospital, physician, or other care-giver, for a service or treatmentprovided to a patient. For example, a first entity, e.g., an insurancecompany, can use the outcome of a method described herein to determinewhether to continue, discontinue, enroll an individual in an insuranceplan or program, e.g., a health insurance or life insurance plan orprogram.

In one aspect, the disclosure features a method of providing data. Themethod includes providing data described herein, e.g., generated by amethod described herein, to provide a record, e.g., a record describedherein, for determining if a payment will be provided. In someembodiments, the data is provided by computer, compact disc, telephone,facsimile, email, or letter. In some embodiments, the data is providedby a first party to a second party. In some embodiments, the first partyis selected from the subject, a healthcare provider, a treatingphysician, a health maintenance organization (HMO), a hospital, agovernmental entity, or an entity which sells or supplies the drug. Insome embodiments, the second party is a third party payor, an insurancecompany, employer, employer sponsored health plan, HMO, or governmentalentity. In some embodiments, the first party is selected from thesubject, a healthcare provider, a treating physician, an HMO, ahospital, an insurance company, or an entity which sells or supplies thedrug and the second party is a governmental entity. In some embodiments,the first party is selected from the subject, a healthcare provider, atreating physician, an HMO, a hospital, an insurance company, or anentity which sells or supplies the drug and the second party is aninsurance company.

In another aspect, the disclosure features a record (e.g., computerreadable record) which includes a list and value of characteristic,e.g., size, sequence, composition or amount for the marker or marker setfor a patient. In some embodiments, the record includes more than onevalue for each marker.

The present invention will now be illustrated by the following Examples,which are not intended to be limiting in any way.

EXAMPLES Example 1 Cell Line Panel Screens

To support clinical development and identify potential biomarkers oftumor sensitivity or resistance, two large cancer cell line panels(Panel 1, N=653 (McDermott et al. (2007) PNAS 104:19936-19941); Panel 2,N=240 (O'Day et al. (2010) Fourth AACR International Conference onMolecular Diagnostics in Cancer Therapeutic Development)) were treatedwith MLN4924 and cell viability data (IC50, EC50, and POC—Percentage ofControl) were generated.

Panel 1 (McDermott et al., supra). The cell lines were exposed to threeMLN4924 concentrations (20 nM, 200 nM, and 2 μM) for 72 hours.Viability, i.e., cell number, was quantified by measuring fluorescenceof a cell-permeant nucleic acid stain. Mean of triplicate values foreach sample were taken and compared to DMSO control to calculatepercentage of control. In the results, control or no activity value isgiven a value of about 1, sensitivity is indicated by a value less than1, with 0 as death of the entire cell population and resistanceindicated by a value greater than 1. A continuum of viability values wasobtained for each concentration, so some values were selected ascut-offs for final determination of sensitivity or resistance. Forexample, median POC values of less than the median value of all POC'srecorded in the panel (<0.34) indicated sensitivity, values of 0.34 to0.75 indicated borderline sensitivity, values greater than the 3^(rd)quartile of all POC's in the panel (>0.75) insensitivity or resistance.In general, a dose response relationship was observed with sensitivecell lines. Final judgment of cell line as sensitive, insensitive orresistant was determined by its viability at 2 μM.

Panel 2 (Ricerca Biosciences, Inc., Bothell, Wash.). MLN4924 was addedin half-log dilutions for 10 concentrations and treated for 72 hours.High-content cell screening by fluorescence microscopy included imageanalysis to generate several types of data. Results included EC50 values(after measurement of cell numbers, the EC50 concentration wascalculated from the inflection point of a curve of percent of control(POC) against log of MLN4924 concentration), IC50 values (from thePOC-log MLN4924 plot, IC50 is the concentration at 50% maximal possibleresponse), apoptosis (measurement of activation of caspase 3 plottedagainst log of MLN4924 concentration, determined as the concentrationfor >5 fold induction), and mitotic activity (determined by measuringthe fold increase of phospho-histone 3). A comparison of EC50 to IC50(FIG. 3) allowed assignment of cell lines to sensitive, insensitive orresistant. Final identification of a cell line as sensitive or resistantwas based on the EC50 values. The cutoff for sensitivity is a medianEC50 of less than the median value of all POC's recorded in the panel(<0.36), borderline sensitivity was associated with median EC50 of 0.36to 1.67 and insensitive or resistant cell lines were identified by EC50greater than the 3^(rd) quartile of all POC's in the panel (>1.67).

Overlapping cell lines between the panels 1 and 2 (114 overlaps) showedconsistent growth inhibition effects (Spearman Correlationcoefficient=0.72). In addition, histology and mutation analysis on eachcell line panel as a whole (not just overlapping cell lines), alsogenerated consistent observations between the two panels (FIGS. 4A andB).

Fisher's Exact Test using median percentage of control values was usedto evaluate associations of individual mutations in the cell lines toMLN4924 sensitivity or resistance. See Table 3 for a summary of p valuesfor selected genes. Genes whose mutations were linked to sensitivity inthe Fisher Exact test include NF2, SMAD4, KDM6A, CDKN2A and CDKN2A_p14.RB1 and TP53 were linked to insensitivity.

TABLE 3 Confidence of mutated marker association with response toMLN4924 Number Number Total N sensitive Panel 1 Total N sensitive Panel2 Mutated Gene panel 1 panel 1 p-value panel 2 panel 2 p-value PhenotypeNF2 10 8 0.067 5 4 0.187 Sensitive SMAD4 24 15 0.197 11 6 0.509Sensitive KDM6A 6 6 0.019 4 4 0.062 Sensitive RB1 32 12 0.062 14 17 1.00Resistant* TP53 235 112 0.013 98 45 0.107 Resistant CDKN2A 153 94 0.00168 38 0.146 Sensitive CDKN2A_p14 114 70 0.01 59 33 0.178 Sensitive*denotes phenotype for RB1 association not conclusive across all celltypes. Some tumor types were more associated with resistance thanothers: tumors from brain, bladder, bone and lung (NSCLC) have p-valuesof 0.102, 0.205, 0.226, and 0.281, respectively. The result that RB1 isnot a sensitivity marker agrees with the result of Jia et al. ((2011)Neoplasia 13: 561-569) which excluded the involvement of RB1 in MLN4924mechanism.

Example 2 Analysis of Mutation Associations

One difficulty with correlating mutations of genes in cell lines withsensitivity to a therapeutic agent is that many cell lines have morethan one mutated gene. For example, cell line named 8505C from thyroidcarcinoma has mutations in BRAF, TP53, NF2 and CDKN2A. In particular,TP53 and CDKN2A mutations co-occurred with other mutations in the celllines. To learn which mutant is associated with sensitivity orresistance in the cell line panels, sub-analyses were performed.

APC vs TP53.

In cell line panel 1, 23 cell lines have a mutation in APC. Of these, 18cell lines also have a mutation in TP53. It was difficult to determinewhether APC is a driver of resistance to MLN4924, or just a passengermutation found frequently in TP53 mutants. Further analysis of the celllines was undertaken by subtraction of cell lines with double mutantswhich included TP53 (Table 4).

TABLE 4 Comparison of TP53 mutant cell lines with APC and other mutantcell lines in panel 1 A. Subtract TP53 mutants Gene sens(78) res(51)p-value RB1 0 2 0.154433 NRAS 7 7 0.285024 APC 2 3 0.30657 SMAD4 2 30.30657 BRCA2 0 1 0.395349 FAM123B 0 1 0.395349 MAP2K4 0 1 0.395349 B.Subtract APC mutants Gene sens(184) res(158) p-value TP53 108 1100.0234828 RB1 12 19 0.0573067 MAP2K4 2 5 0.1665217 C. Double MutantsGene sens(190) res(175) p-value APC + TP53 4 14 0.00842

As can be seen in Table 4A, subtracting TP53 mutants from the cell linepanel leaves an N too small to allow conclusion of association ofremaining mutations with resistance of the TP53 wt cell lines totreatment with MLN4924. After removing all 23 APC mutants, TP53 stillappears to be associated with resistance (Table 4B). Nevertheless, celllines with both APC and TP53 mutations show strong association toresistance (Table 4C). Additionally, a majority of the cell lines in theAPC+TP53 mutant subgroup are from intestinal cancer tumor samples (Table5, which also includes cell lines and data from panel 2 and six panel 1cell lines which were not included in the original subtractiveanalysis).

TABLE 5 Subset of cell lines with mutations in APC and TP53 TumorViability Tissue Tumor at 2 μM EC50 Co-occurring Cell line SourceHistology Panel 1 Panel 2 mutations HT55 intestine colon carcinoma0.5143 APC:APC:TP53 SW 1116 intestine colon 1.1599 10APC:APC:KRAS:SMAD4:TP53 adenocarcinoma COCM1 intestine colon carcinoma1.1438 APC:APC:PIK3CA:SMAD4:TP53 LS1034 intestine adenocarcinoma 3.76APC:KRAS:TP53 SW 1463 intestine rectum 0.8963 TP53:FBXW7:KRAS:APCadenocarcinoma NCI-H630 intestine colorectal 0.75 APC:TP53adenocarcinoma SW1417 intestine colon 0.6374 9.03 APC:TP53:BRAF:PIK3R1adenocarcinoma SW837 intestine rectum 0.702 0.694 APC: APC:adenocarcinoma FAM123B:TP53:FBXW7:KRAS SW620 intestine colon 0.49280.387 APC:TP53:TP53:KRAS:MAP2K4: adenocarcinoma SMAD4 NCI-H1581 lungsquamouscell 0.7365 APC:TP53 NSCLC carcinoma HCT-15 intestine colon0.6654 0.836 APC:BRCA2:FAM123B:KRAS:MSH6: adenocarcinoma PIK3CA: TP53C2BBe1 intestine colorectal 0.7159 APC:TP53 adenocarcinoma T84 intestinecolon carcinoma 0.5777 0.556 KRAS:PIK3CA:TP53:APC SW626 ovaryadenocarcinoma 0.5541 TP53:KRAS:APC HT29 intestine colorectal 0.43760.25 APC:PIK3CA:TP53:BRAF:SMAD4:APC adenocarcinoma LK-2 lung lung NSCLC0.2893 TP53:APC:CDKN2A MKN28 stomach metastasis 0.2847 APC:TP53:NF1COLO-205 intestine colon 0.144 0.0923 TP53:BRAF:APC:SMAD4 adenocarcinomaHGC-27 stomach gastric 0.1291 TP53:APC:PTEN:PIK3CA carcinoma NCIH747intestine cecum 2.07 APC:APC:KRAS:TP53 adenocarcinoma SKUT1 smoothleiomyosarcoma 0.0856 APC:APC:PIK3CA:PTEN:PTEN:RB1: muscle TP53:TP53SK-MEL-30 skin malignant 0.7728 APC:NRAS:TP53 melanoma COLO320 intestinecolon 1.78 APC:TP53 HSR adenocarcinoma NCI-H520 lung squamouscell 0.55770.261 CDKN2A:APC:TP53 NSCLC carcinoma NCI-H1703 lung adenosquamous0.1101 TP53:APC:CDKN2A NSCLC carcinoma NCI-H1975 lung adenocarcinoma0.3049 TP53:APC:CDKN2A:PIK3CA:EGFR RCM-1 intestine rectum 0.7666TP53:KRAS:APC adenocarcinoma SK-OV-3 ovary adenocarcinoma 0.397 10TP53:PIK3CA:CDKN2A:APC

Association with Histology.

It is possible that some TP53 mutations associate with resistance insome types of tumor more than others. TP53 mutant cell lines wereanalyzed within tissue types by a Mann-Whitney (nonparametric) test. Ascan be seen in FIG. 5, TP53 mutations are significantly correlated withresistance to MLN4924 in colon cancer cell lines (p-value=0.04022).

Similar analysis of associations in other tissues indicates that TP53mutation is associated with MLN4924 resistance in breast cancer and lungcancer (NSCLC) cell lines.

Additional mutations were analyzed for possible association of tumorhistology with resistance or sensitivity to MLN4924 treatment. Table 6provides results from analysis of panel 1 and Table 7 provides resultsfrom analysis of panel 2, whose smaller size proved a challenge for thistype of analysis. The cutoff of association with the tissues was chosenat p-values of <0.05.

TABLE 6 Association of mutation with histology in Panel 1 Tumor Nmutants N wild N mutants N wild Tissue this type this all type allSource Mutation cancer cancer cancers cancers Association p-valueHead_Neck TP53 15 1 111 79 sensitive 0.0001 Head_Neck CDKN2A 12 4 94 96sensitive 0.0072 Head_Neck SMAD4 3 13 15 175 sensitive 0.0262 CervixTP53 1 6 124 51 resistant 0.0027 Lung RB1 2 4 19 156 resistant 0.0178Bone RB1 1 5 12 178 sensitive 0.0477

TABLE 7 Association of mutation with histology in Panel 2 Tumor Nmutants N wild N mutants N wild Tissue this type this all type allSource Mutation cancer cancer cancers cancers Association p-value skinCDKN2A 8 1 30 43 resistant 0.0022 skin CDKN2A.p14 6 3 26 47 resistant0.0004 skin TP53 2 7 53 20 resistant 0.0036 CNS CDKN2A.p14 5 4 26 47resistant 0.0466* *p-value for CNS derived from percent of controlviability. For skin, p-value derived from EC50.

In contrast to the general association of TP53 with resistance toMLN4924, in head and neck cancer, TP53 mutation is associated withsensitivity. A similar contrast was found for CDKN2A mutations in skinand central nervous system (CNS). CDKN2A or CDKN2A.p14 mutations wereassociated with resistance to MLN4924 treatment in skin and CNS tumorcell lines, despite the general association with CDKN2A mutation andsensitivity to MLN4924 treatment. Tables 6 and 7 also show that TP53mutations are significantly associated with resistance in cervicalcancer and skin cancer; CDKN2A mutations are significantly associatedwith sensitivity in head and neck cancer; SMAD4 mutations aresignificantly associated with sensitivity in head and neck cancer; RB1mutations are significantly associated with resistance in lung cancer;and RB1 mutations are significantly associated with sensitivity in bonecancer.

Example 3 Individual Cell Line Screening Results

The following tables include results of the individual cell line screenswhich led to conclusions about markers whose mutations confersensitivity to MLN4924. Notation of the mutations and explanation ofmutation syntax can be found in the COSMIC database.

TABLE 8 Results of screens of cell lines with mutations in NF2.Viability EC50 ORF mutation Protein mutation Tumor Tissue Tumor at 2 μMPanel Co-occurring Cell line (SEQ ID NO: 2) (SEQ ID NO: 3) Source typePanel 1 2 Phenotype mutations 647-V c.115-1G > C p.? urinary_tractprimary 0.038 0.283 sensitive MAP2K4: NF2: RB1: TP53: TP53 ACHN c.169C >T p.R57* kidney primary 0.0621 0.652 sensitive CDKN2A: CDKN2a(p14): NF2CAL-62 c.643G > T p.E215* thyroid primary 0.0676 0.0837 sensitiveCDKN2A: CDKN2a(p14): KRAS: NF2: TP53 NUGC-3 c.683delA p.K228fs*23stomach primary 0.098 sensitive NF2: TP53 SW1573 c.1_363del363 p.? lungprimary 0.1114 sensitive CDKN2A: CDKN2a(p14): CTNNB1: KRAS: NF2: PIK3CA:SMAD4 8505C c.385G > T p.E129* thyroid primary 0.1405 sensitive BRAF:CDKN2A: NF2: TP53 CaR-1 c.115_1737del1623 p.? large_intestine primary0.3244 sensitive CDKN2A: CDKN2a(p14): NF2: STK11: TP53 SN12C c.115-1G >C p.? kidney primary 0.3357 sensitive NF2: TP53 MDA-MB-231 c.691G > Tp.E231* breast primary 0.5284 0.871 insensitive BRAF: CDKN2A:CDKN2a(p14): KRAS: NF2: TP53 S-117 c.221G > A p.W74* soft_tissue primary0.6412 resistant CDKN2A: CDKN2a(p14): NF2: TP53 RL95-2 c.514delAp.R172fs*2 endometrium primary 0.094 sensitive BRCA2: BRCA2: c.1084C > Tp.Q362* HRAS: NF2: NF2: PTEN: PTEN: TP53: TP53

TABLE 9 Results of screens of cell lines with mutations in SMAD4.Viability EC50 ORF mutation Protein mutation Tumor Tissue Tumor at 2 μMPanel Co-occurring Cell line (SEQ ID NO: 5) (SEQ ID NO: 6) Source typePanel 1 2 Phenotype mutations CAL-27 c.733C > T p.Q245* upper_(—)primary 0.0271 0.139 sensitive CDKN2A: SMAD4: aerodigestive_(—) TP53tract KP-4 c.1_1659del1659 p.0? pancreas metastasis 0.0518 sensitiveCDKN2A: CDKN2a(p14): KRAS: SMAD4 GAMG c.1_249del249 p.? central_(—)primary 0.0721 sensitive CDKN2A: nervous_(—) CDKN2a(p14): system SMAD4:TP53 PANC- c.905_1659del755 p.? pancreas primary 0.0724 sensitiveCDKN2A: 03-27 CDKN2a(p14): KRAS: SMAD4: TP53 FADU c.1_1659del1659 p.0?upper_(—) primary 0.073 0.261 sensitive CDKN2A: SMAD4: aerodigestive_(—)TP53: TP53 tract SW1573 c.1_1659del1659 p.0? lung primary 0.1114sensitive CDKN2A: CDKN2a(p14): CTNNB1: KRAS: NF2: PIK3CA: SMAD4 KYSE-c.788-1G > A p.? oesophagus primary 0.1279 sensitive SMAD4: TP53 150COLO- c.1_667del667 p.? large_intestine primary 0.144 0.0923 sensitiveAPC: BRAF: 205 SMAD4: TP53 CAL-33 c.766C > T p.Q256* upper_(—) primary0.1445 sensitive CDKN2A: PIK3CA: aerodigestive_(—) SMAD4: TP53 tractNCI-N87 c.1_955del955 p.? stomach metastasis 0.1461 sensitive SMAD4:TP53 CAPAN-1 c.1028C > G p.S343* pancreas metastasis 0.1473 0.365sensitive? BRCA2: CDKN2A: CDKN2a(p14): KRAS: SMAD4: TP53 YAPC c.1543delAp.R515fs*22 pancreas primary 0.1871 0.234 sensitive CDKN2A: CDKN2a(p14):KRAS: SMAD4: TP53 NCI- c.1_1659del1659 p.0? lung metastasis 0.2554sensitive BRAF: CDKN2A: H2405 CDKN2a(p14): MAP2K4: SMAD4: TP53 CFPAC-1c.1_1659del1659 p.0? pancreas metastasis 0.2822 6.37 sensitive? KRAS:SMAD4: TP53 MDA- c.1_1659del1659 p.0? breast metastasis 0.2965 0.0267sensitive PTEN: RB1: MB-468 SMAD4: TP53 MKN45 c.1_1659del1659 p.0?stomach metastasis 0.3614 insensitive CDKN2A: CDKN2a(p14): SMAD4 BxPC-3c.1_1659del1659 p.0? pancreas primary 0.4186 0.251 insensitive CDKN2A:CDKN2a(p14): MAP2K4: SMAD4: TP53 HT-29 c.931C > T p.Q311*large_intestine primary 0.4376 0.25 insensitive APC: APC: BRAF: PIK3CA:SMAD4: TP53 UMC-11 c.1606_1612delCTAGACG p.L536fs*14 lung primary 0.4472insensitive CDKN2A: CDKN2a(p14): SMAD4: STK11: TP53 SW620 c.955 + 5G > Cp.? large_intestine primary 0.4928 0.387 insensitive APC: KRAS: MAP2K4:SMAD4: TP53: TP53 PANC- c.366_367insA p.C123fs*2 pancreas primary 0.7767resistant CDKN2A: 08-13 CDKN2a(p14): KRAS: SMAD4 COLO- c.1_1659del1659p.0? large_intestine metastasis 0.814 resistant APC: CDKN2A: 678CDKN2a(p14): FAM123B: KRAS: SMAD4 CoCM-1 c.956_1659del704 p.?large_intestine primary 1.1438 resistant APC: APC: PIK3CA: SMAD4: TP53SW954 c.378_379delCT p.V128fs*14 vulva primary 0.324 sensitive SMAD4:TP53

TABLE 10 Results of screens of cell lines with mutations in KDM6A.Protein mutation Viability EC50 Cell ORF mutation (SEQ ID NO: 10 TumorTissue Tumor at 2 μM Panel line (SEQ ID NO: 8 or 9) or 11) Source typePanel 1 2 Phenotype Co-occurring mutations HCC1806 c.444_564del121 p.0breast primary 0.0116 sensitive CDKN2A: CDKN2a(p14): KDM6A: STK11: TP53KU-19-19 c.2587C > T p.Q863* urinary_tract primary 0.058 sensitiveCDKN2A: CDKN2a(p14): KDM6A: NRAS MIA- c.1_4206del4206 p.0? pancreasprimary 0.1145 0.239 sensitive CDKN2A: CDKN2a(p14): PaCa-2 KDM6A: KRAS:TP53 KYSE- c.385_654del270 p.0 oesophagus primary 0.1426 sensitiveCDKN2A: CDKN2a(p14): 450 EGFR: KDM6A: NOTCH1: TP53: TP53 KYSE- c.997C >T p.Q333* oesophagus primary 0.2154 sensitive CDKN2A: CDKN2a(p14): 180KDM6A: TP53 LS-174T c.3945_3946insA p.E1316fs* large_intestine primary0.3262 0.137 sensitive CTNNB1: KDM6A: KRAS: 17 PIK3CA BV-173c.226_384del159 p.0 haematopoietic, primary 0.131 sensitive CDKN2A:CDKN2a(p14): lymphoid_tissue KDM6A THP-1 c.1_1923del1923 p.0haematopoietic, primary 0.148 sensitive CDKN2A: CDKN2a(p14):lymphoid_tissue KDM6A: NRAS: TP53

TABLE 11 Sampling of results of screens of cell lines with mutations inFBXW7. Viability EC50 Cell ORF mutation Protein mutation Tumor TissueTumor at 2 μM Panel line (SEQ ID NO: 13) (SEQ ID NO: 14) Source typePanel 1 2 Phenotype Co-occurring mutations AN3CA c.1321C > T p.R441WUterus metastasis 0.1944 sensitive PTEN: TP53: PIK3R1 ESS-1 c.1393C > Tp.R465C Uterus primary 0.0529 sensitive FBXW7: TP53: PIK3CA: RB1 C-33ac.1394G > A p.R465H Cervix primary 0.0822 sensitive RB1: TP53: MSH2:PIK3CA: PTEN HuCCT1 c.881C > G p.S294* Liver primary 0.1262 sensitiveTP53: KRAS MKN1 c.1393C > T p.R465C Stomach metastasis 0.4631insensitive TP53: PIK3CA AsPC-1 c.1393C > T p.R465C Pancreas metastasis0.9889 resistant CDKN2A: MAP2K4: KRAS: TP53 RCM-1 c.1513C > T p.R505CIntestine primary 0.7666 resistant TP53: KRAS SW1463 c.1436G > A p.R479QIntestine primary 0.8963 resistant TP53: KRAS: APC

Example 4 Association of TP53 Deletion with Resistance

Another approach to determine the role of TP53 in responsiveness toMLN4924 was a study in which the TP53 gene was deleted. In earlierstudies, the importance of p53 in the rereplication response to MLN4924seemed to be dependent on the specific genetic manipulation and wasexpected to closely mirror that of CDT1 overexpression (Cdt1 is asubstrate of two alternative CRL complexes and is stabilized by MLN4924in many cell lines). In knockdown studies, p53 appeared to behavesimilarly to CDT1 knockdown at early timepoints, but not latertimepoints, unless higher concentrations of MLN4924 were used. Westernblotting suggested efficient p53 protein knockdown by the siRNASMARTpool, although RNAi generally does not result in the complete lossof protein. Therefore, the residual protein may still affect theresponse to MLN4924, particularly since MLN4924 results in thestabilization of p53 (Liao et al. (2011) Mol. Cell Proteomics10:10.1074/mcp.M111.009183). The viability effect of MLN4924 wasassessed on HCT-116 cells that were genetically deleted for p53 togetherwith their parental control.

Paired isogenic HCT-116 cell lines that were either wild-type (+/+) ornull (−/−) for p53 expression (HD PAR-018 and HD 104-001, respectively,Horizon Discovery Ltd) were seeded in separate 384-well plates and thentreated the following day with a titration of MLN4924 in triplicate, andincubated for 24, 36, 48 or 72 h, with seeding densities of 1600, 1200,800, and 400 cells/well, respectively. Following compound incubation,viability of HCT-116 cells was assessed by ATPlite assay (Perkin Elmer)according to the manufacturer's instructions using the LEADseekerimaging system (GE Healthcare).

HCT-116 TP53+/+ cells (MLN4924 LC₅₀=21±1 nM) demonstrated greaterMLN4924 sensitivity at 72 h than HCT-116 TP53−/− cells (MLN4924LC₅₀=74±5 nM; FIGS. 6A-D). These results suggest that p53 deficiencymakes HCT-116 less sensitive to MLN4924, suggesting that the overarchingrole of p53 at 72 h is proapoptotic. Earlier time points reinforce thisinterpretation, as TP53−/− cells have less cell death at the highestdrug concentrations at 24, 36, and 48 h. Western blots showed that p21was still up-regulated by MLN4924 in TP53−/− HCT-116 cells. In HCT-116cells, the stabilization of p21 may be a direct effect of inhibition ofCRL4-Cdt2 (Nishitani et al., 2008; Abbas et al., 2008; Kim et al.,2008).

This result is contrary to the conclusion in Lin et al. (2010) CancerRes. 70:10310-10320) using the HCT116−/− p53 knockout cells. In thatstudy, it was concluded that the TP53 knockout cells were moresusceptible to overall cell death or growth inhibition by MLN4924. Thereason the present study comes to a different conclusion than in Lin etal. is the amount of time the cells were treated with MLN4924. In Lin etal, the cells were treated for 8 hours before a washout. In the presentstudies and in the cell line panels of the earlier examples, the cellswere treated with MLN4924 continuously over 72 hours. In the washout,the p53 levels are allowed to stabilize and take advantage of activatingalternative pathways than the earlier pathways which were initiallyinhibited and led to the earlier susceptibility.

Example 5 Isolation of Nucleic Acid and Nucleic Acid Sequencing Methods

Genomic isolations and DNA sequencing. DNA isolation from cells andtumors is conducted using DNAEASY® isolation kit (Qiagen, Valencia,Calif.). RNA isolation is conducted using MegaMax (Ambion division ofApplied Biosystems, Austin, Tex.). Genomic isolations are conductedfollowing manufacturer recommend protocols.

SANGER Sequencing Methodology.

PCR amplifications are conducted using optimized cycling conditions pergene-exon. Primer extension sequencing is performed using AppliedBiosystems BigDye version 3.1. The reactions are then run on AppliedBiosystem's 3730xl DNA Analyzer. Sequencing base calls are done usingKB™ Basecaller (Applied Biosystems). Somatic Mutation calls aredetermined by Mutation Surveyor (SoftGenetics) and confirmed manually byaligning sequencing data with the corresponding reference sequence usingSeqman (DNASTAR).

SEQUENOM Sequencing Methodology.

Sequenom (San Diego, Calif.) assays are designed using TypePLEX®chemistry with single-base extension. This process consists of threesteps: 1) A text file containing the SNPs or mutations of interest andflanking sequence is uploaded at mysequenom.com where it is run througha web based program ProxSNP, 2) The output of ProxSNP is run throughPreXTEND and 3) the output of PreXTEND is run through Assay Design whichdetermines the expected mass weight of the extend products to ensureseparation between all potential peaks found within a multiplexedreaction.

PCR primers are then designed to bracket the region identified in theassay design steps. The region of interest is amplied in PCR reactionsusing the primers. 15 nl of amplified and extended product is spotted ona 384 SpectroCHIP II using a Nanodispenser RS1000. A 3-point calibrantis added to every chip to ensure proper performance of the SequenomMaldi-tof compact mass spectrometer.

The SpectroCHIP II is placed in the Sequenom MALDI-TOF compact massspectrometer. The mass spectrometer is set to fire a maximum of 9acquisitions for each spot on the 384 well SpectroCHIP. TypePLEX Goldkit SpectroCHIP II from Sequenom (10142-2) is used followingmanufacturers recommended protocols. Analysis is performed usingSequenom analysis software, MassARRAY® Typer Analyzer v4.

NEXT GENERATION SEQUENCING (NGS) Methodology.

Targeted NGS using the Illumina platform (Illumina, Inc. San Diego,Calif.) is used to confirm and identify low frequency mutations in amarker. Primer pairs are designed to amplify coding exons. PCR productsare quantified using a PicoGreen assay and combined in equal molarratios for each sample. The purified products are end-repaired andconcatenated by ligation. The concatenated products are used for Hi-Seq2000 library preparation. The concatenated PCR products are sheared andused to make barcoded Hi-Seq 2000 libraries consisting of 12 barcodedsamples per multiplexed pool. The pooled Hi-Seq 2000 libraries undergoclonal amplification by cluster generation on eight lanes of a Hi-Seq2000 flow cell and are sequenced using 1×100 single-end sequencing on aHi-Seq 2000. Matching of primary sequencing reads to the human genomebuild Hg18, as well as SNP analysis are performed using Illumina'sCASAVA software version 1.7.1.

General Procedures Quantitative RT-PCR

cDNA synthesis and quantitative RT-PCR is performed using ABI GeneExpression Assays, reagents, and ABI PRISM® 7900HT Sequence DetectionSystems (Applied Biosystems, Foster City, Calif.) using the followingcycle conditions: hold at 50° C. for 2 minutes for AmpErase UNGactivation, then 95.0° C. for 10 minutes to activate DNA polymerase thenrun 40 two-part cycles of 95.0° C. for 15 seconds and 60.0° C. for 1minute. The dCt is calculated by using the average Ct of control genesB2M (Hs99999907_ml) and RPLPO (Hs99999902_ml). Relative mRNA expressionquantification is derived using the Comparative Ct Method (AppliedBiosystems). mRNA expression fold change values are generated from anormal sample and corresponding tumor sample.

Sample Handling for Myeloma Samples

Upon collection of patient bone marrow aspirate, the myeloma cells areenriched via rapid negative selection. The enrichment procedure employsa cocktail of cell-type specific antibodies coupled with an antibodythat binds red blood cells RosetteSep (Stem Cell Technologies). Theantibody cocktail has antibodies with the following specificity: CD14(monocytes), CD2 (T and NK cells), CD33 (myeloid progenitors andmonocytes), CD41 (platelets and megakaryocytes), CD45RA (naïve B and Tcells) and CD66b (granulocytes). The antibodies cross-link thenon-myeloma cell types to the red blood cells in the samples. The boundcell types are removed using a modified ficoll density gradient. Myelomacells are then collected and frozen.

Total RNA is isolated using a QIAGEN® Group RNEASY® isolation kit(Valencia, Calif.) and quantified by spectrophotometry.

DNA is isolated from the flow through fraction of the column used in theRNA isolation method.

Analysis of Myeloma Gene Expression on an Array

RNA is converted to biotinylated cRNA by a standard T7 basedamplification protocol (AFFYMETRIX® Inc., Santa Clara, Calif.). A smallnumber of samples with ≧0.5-2.0 μg are also labeled and subsequentlyhybridized if 6 μg of cRNA is produced. For the automated T7amplification procedure, the cDNA and the biotin labeled cRNA arepurified using AMPURE® PCR Purification System, following themanufacturer's protocol (AGENCOURT® Bioscience Corporation, Beverly,Mass.). The cRNA yield is assessed by spectrophotometry and 10 μg ofcRNA is fragmented and further processed for triplicate hybridization onthe AFFYMETRIX® Human Genome HG-U133A and HG-U133B GENECHIP® arrays. Incases where cRNA yield ranged between 6 μg to 10 μg, the entire cRNAsample is fragmented.

cRNA for each sample is hybridized to the U133A/B arrays in triplicate;operators, chip lots, clinical sites and scanners (GENECHIP® Scanner3000) are controlled throughout. Background subtraction, smoothingadjustment, noise corrections, and signal calculations are performedwith AFFYMETRIX® MAS5.0 Quality control metrics include: percent presentcall (>25) scale factor (<11), β-actin 3′:5′ ratio (<15) and background(<120). Samples that fall outside these metrics are excluded fromsubsequent analysis.

The myeloma purity score examines expression of genes known in theliterature to be expressed highly in myeloma cells (and their normalplasma precursor cells), to expression of genes known to be expressedhighly in erythroid cells, neutrophils and T cells—see list of 14markers below). The myeloma score=expression of myeloma markers (#1-4below)/erythroid (#5-7)+neutrophil (#8-11)+T cell (#12-14):

1. 205692_s_at CD38 CD38 antigen (p45) myelomalplasma cell2. 201286_at SDC1 syndecan-1 myelomalplasma cell3. 201891_s_at B2M beta-2 microglobulin myeloma/plasma cell4. 211528_x_at B2M beta-2 microglobulin myelomalplasma cell5. 37986_at EpoR erythropoetin receptor erythroid cell6. 209962_at EpoR erythropoetin receptor erythroid cell7. 205838_at GYPA glycophorinA erythroid cell8. 203948_s_at MPO myeloperoxidase neutrophil9. 203591_s_at CSFR3 colony stimulating factor 3 receptor (granulocyte)neutrophil10. 204039_at CEBPACCAAT/enhancer bindingprotein (C/EBP), alphaneutrophil11. 214523_at CEBPECCAAT/enhancer bindingprotein (C/EBP), epsilonneutrophil12. 209603_at GATA3 GATA binding protein 3 T lymphocyte13. 209604_s_at GATA4 GATA binding protein 4 T lymphocyte14. 205456_at CD3ECD3E antigen, epsilon polypeptide T lymphocyteSamples with a myeloma purity score less than 10 are excluded fromfurther analysis.

EQUIVALENTS

Although embodiments of the invention have been described using specificterms, such description are for illustrative purposes only, and it is tobe understood that changes and variations may be made without departingfrom the spirit or scope of the invention. Those skilled in the art willrecognize, or be able to ascertain using no more than routineexperimentation, many equivalents of the specific embodiments of theinvention described herein. Such equivalents are intended to beencompassed by the following claims.

1. A method for identifying a cancer patient for treatment with aNEDD8-activating enzyme (NAE) inhibitor comprising: a) measuring atleast one characteristic of at least one marker associated with at leastone marker gene in a patient sample comprising tumor cells, wherein theat least one characteristic is selected from the group consisting ofsize, sequence, composition and amount; b) identifying whether the atleast one characteristic measured in step a) is informative for outcomeupon treatment with the NAE inhibitor, wherein the NAE inhibitor is((1S,2S,4R)-4-{4-[(1S)-2,3-dihydro-1H-inden-1-ylamino]-7H-pyrrolo[2,3-d]pyrimidin-7-yl}-2-hydroxycyclopentyl)methylsulphamate; and c) identifying the patient for treatment with the NAEinhibitor if the informative characteristic indicates that the tumorcells comprise at least one marker gene whose mutational statusindicates a favorable outcome to NAE inhibition therapy, wherein the atleast one marker gene is a tumor suppressor with a relationship to theactivity of a cullin ring ligase and the mutational status is mutant.2.-3. (canceled)
 4. The method of claim 1, wherein the at least onemarker gene is selected from the group consisting of NF2, SMAD4, KDM6A,and FBXW7.
 5. The method of claim 1, wherein the mutation in the atleast one marker gene is an inactivating mutation.
 6. (canceled)
 7. Themethod of claim 1, wherein the at least one marker is selected from thegroup consisting of nucleic acid and protein corresponding to the atleast one marker gene.
 8. The method of claim 1, wherein the patientsample comprises hematological tumor cells or solid tumor cells. 9.(canceled)
 10. The method of claim 1, wherein the at least one marker isat least two markers.
 11. The method of claim 1, wherein the at leastone characteristic is sequence of at least one marker. 12.-14.(canceled)
 15. The method of claim 1, further comprising determiningwhether to continue NAE inhibitor treatment of cancer in a patientcomprising: a) obtaining a second biological sample comprising tumorcells from the patient, wherein the patient is treated with an NAEinhibitor prior to the second sample; b) measuring the at least onecharacteristic of at least one marker in the second sample; c) comparingthe results of the measurements in b) with the results of the firstsample; and d) determining to continue treatment with the NAE inhibitorif the comparison indicates that the tumor cells in the second samplecomprise at least one marker gene whose mutational status indicates afavorable outcome, wherein the at least one marker gene is a tumorsuppressor with a relationship to the activity of a cullin ring ligase.16.-26. (canceled)
 27. A kit comprising a reagent to measure at leastone characteristic of at least one marker in a patient sample, whereinthe at least one characteristic is selected from the group consisting ofsize, sequence, composition and amount, wherein the at least one markercorresponds to at least one marker gene is a tumor suppressor with arelationship to the activity of a cullin ring ligase.
 28. (canceled) 29.The kit of claim 27, wherein the at least one marker is selected fromthe group consisting of nucleic acid and protein associated with the atleast one marker gene.
 30. The kit of claim 27, wherein the at least onemarker gene is selected from the group consisting of NF2, SMAD4, KDM6A,and FBXW7.
 31. The kit of claim 27, further comprising a stabilizer toadd to the sample.
 32. The kit of claim 29, wherein the at least onemarker is nucleic acid and the reagent is at least one primer.
 33. Thekit of claim 32, wherein the at least one primer hybridizes to a nucleicacid sequence selected from the group consisting of SEQ ID NO: 1, 2, 4,5, 7, 8, 9, 12, 13, a sequence on chromosome 22q from base pair 29999545to 30094589, sequence on chromosome 18q from base pair 48556583 to48611412, sequence on chromosome Xp from base pair 44732423 to 44971847,sequence on chromosome 4q from base pair 153242410 to 153456172, and acomplement of any of the foregoing. 34.-35. (canceled)
 36. The method ofclaim 8, wherein the patient sample comprising hematological tumor cellsis blood.
 37. The method of claim 36, further comprising enriching thepatient sample for tumor cells. 38.-39. (canceled)
 40. A method forpaying for the treatment of cancer with an NAE inhibitor comprising: a)recording the mutational status of at least one marker gene in a patientsample comprising tumor cells, and b) authorizing payment of the NAEinhibitor treatment if the mutational status indicates a favorableoutcome, wherein the at least one marker gene is selected from the groupconsisting of NF2, SMAD4, KDM6A, and FBXW7 and the mutational status ismutant. 41.-42. (canceled)
 43. A method for treating a cancer patientwith a therapeutic regimen comprising an NAE inhibitor, comprising: a)using the mutational status of at least one marker gene of a patient'stumor, wherein the at least one marker gene is selected from the groupconsisting of NF2, SMAD4, KDM6A and FBXW7, to select for treatment apatient who is expected to have a favorable outcome with the therapeuticregimen, and b) treating the cancer patient with the NAE inhibitor,wherein the NAE inhibitor is a 1-substituted methyl sulfamate.
 44. Themethod of claim 43, wherein the NAE inhibitor is((1S,2S,4R)-4-{4-[(1S)-2,3-dihydro-1H-inden-1-ylamino]-7H-pyrrolo[2,3-d]pyrimidin-7-yl}-2-hydroxycyclopentyl)methylsulphamate.